Recombinant production of novel polyketides

ABSTRACT

Novel polyketides and novel methods of efficiently producing both new and known polyketides, using recombinant technology, are disclosed. In particular, a novel host-vector system is described which is used to produce polyketide synthases which in turn catalyze the production of a variety of polyketides.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation-in-part of U.S. patentapplication Ser. No. 08/238,811, filed May 6, 1994, which is acontinuation-in-part of U.S. patent application Ser. No. 08/164,301,filed Dec. 8, 1993, now abandoned, which is a continuation-in-part ofU.S. application Ser. No. 08/123,732, filed Sep. 20, 1993, from whichpriority is claimed pursuant to 35 U.S.C. §120, and which disclosuresare hereby incorporated by reference in their entireties.

REFERENCE TO GOVERNMENT CONTRACT

[0002] This invention was made with United States Government support inthe form of a grant from the National Science Foundation (BCS-9209901).

TECHNICAL FIELD

[0003] The present invention relates generally to polyketides andpolyketide syntheses. In particular, the invention pertains to therecombinant production of polyketides using a novel host-vector system.In addition, the invention relates to the combinatorial biosynthesis ofpolyketides.

BACKGROUND OF THE INVENTION

[0004] Polyketides are a large, structurally diverse family of naturalproducts. Polyketides possess a broad range of biological activitiesincluding antibiotic and pharmacological properties. For example,polyketides are represented by such antibiotics as tetracyclines anderythromycin, anticancer agents including daunomycin,immunosuppressants, for example FK506 and rapamycin, and veterinaryproducts such as monensin and avermectin. Polyketides occur in mostgroups of organisms and are especially abundant in a class of mycelialbacteria, the actinomycetes, which produce various polyketides.

[0005] Polyketide synthases (PKSs) are multifunctional enzymes relatedto fatty acid synthases (FASs). PKSs catalyze the biosynthesis ofpolyketides through repeated (decarboxylative) Claisen condensationsbetween acylthioesters, usually acetyl, propionyl, malonyl ormethylmalonyl. Following each condensation, they introduce structuralvariability into the product by catalyzing all, part, or none of areductive cycle comprising a ketoreduction, dehydration, andenoylreduction on the β-keto group of the growing polyketide chain. PKSsincorporate enormous structural diversity into their products, inaddition to varying the condensation cycle, by controlling the overallchain length, choice of primer and extender units and, particularly inthe case of aromatic polyketides, regiospecific cyclizations of thenascent polyketide chain. After the carbon chain has grown to a lengthcharacteristic of each specific product, it is released from thesynthase by thiolysis or acyltransfer. Thus, PKSs consist of families ofenzymes which work together to produce a given polyketide. It is thecontrolled variation in chain length, choice of chain-building units,and the reductive cycle, genetically programmed into each PKS, thatcontributes to the variation seen among naturally occurring polyketides.

[0006] Two general classes of PKSs exist. One class, known as Type IPKSs, is represented by the PKSs for macrolides such as erythromycin.These “complex” or “modular” PKSs include assemblies of several largemultifunctional proteins carrying, between them, a set of separateactive sites for each step of carbon chain assembly and modification(Cortes, J. et al. Nature (1990) 348:176; Donadio, S. et al. Science(1991) 252:675; MacNeil, D. J. et al. Gene (1992) 115:119). Structuraldiversity occurs in this class from variations in the number and type ofactive sites in the PKSs. This class of PKSs displays a one-to-onecorrelation between the number and clustering of active sites in theprimary sequence of the PKS and the structure of the polyketidebackbone.

[0007] The second class of PKSs, called Type II PKSs, is represented bythe synthases for aromatic compounds. Type II PKSs have a single set ofiteratively used active. sites (Bibb, M. J. et al. EMBO J. (1989)8:2727; Sherman, D. H. et al. EMBO J. (1989) 8:2717; Fernandez-Moreno,M. A. et al. J. Biol. Chem. (1992) 267:19278).

[0008] In contrast, fungal PKSs, such as the 6-methylsalicylic acid PKS,consist of a single multi-domain polypeptide which includes all theactive sites required for the biosynthesis of 6-methylsalicylic acid(Beck, J. et al. Eur. J. Biochem. (1990) 192:487-498; Davis, R. et al.Abstr. of the Genetics of Industrial Microorganism Meeting, Montreal,abstr. P288 (1994)).

[0009] Streptomyces is an actinomycete which is an abundant producer ofaromatic polyketides. In each Streptomyces aromatic PKS so far studied,carbon chain assembly requires the products of three open reading frames(ORFs). ORF1 encodes a ketosynthase (KS) and an acyltransferase (AT)active site; ORF2 encodes a PKS chain length determining factor (CLF);and ORF3 encodes a discrete acyl carrier protein (ACP).

[0010]Streptomyces coelicolor produces the blue-pigmented polyketide,actinorhodin. The actinorhodin gene cluster (act), has been cloned(Malpartida, F. and Hopwood, D. A. Nature (1984) 309:462; Malpartida, F.and Hopwood, D. A. Mol. Gen. Genet. (1986) 205:66) and completelysequenced (Fernandez-Moreno, M. A. et al. J. Biol. Chem. (1992)267:19278; Hallam, S. E. et al. Gene (1988) 74:305; Fernandez-Moreno, M.A. et al. Cell (1991) 66:769; Caballero, J. et al. Mol. Gen. Genet.(1991) 230:401). The cluster encodes the PKS enzymes described above, acyclase and a series of tailoring enzymes involved in subsequentmodification reactions leading to actinorhodin, as well as proteinsinvolved in export of the antibiotic and at least one protein thatspecifically activates transcription of the gene cluster. Other genesrequired for global regulation of antibiotic biosynthesis, as well asfor the supply of starter (acetyl CoA) and extender (malonyl CoA) unitsfor polyketide biosynthesis, are located elsewhere in the genome.

[0011] The act gene cluster from S. coelicolor has been used to produceactinorhodin in S. parvulus. Malpartida, F. and Hopwood, D. A. Nature(1984) 309:462. Bartel et al. J. Bacteriol. (1990) 172:4816-4826,recombinantly produced aloesaponarin II using S. galilaeus transformedwith an S. coelicolor act gene cluster consisting of four genetic loci,actI, actIII, actIV and actVII. Hybrid PKSs, including the basic actgene set but with ACP genes derived from granaticin, oxytetracycline,tetracenomycin and frenolicin PKSs, have also been designed which areable to express functional synthases. Khosla, C. et al. J. Bacteriol.(1993) 175:2197-2204. Hopwood, D. A. et al. Nature (1985) 314:642-644,describes the production of hybrid polyketides, using recombinanttechniques. Sherman, D. H. et al. J. Bacteriol. (1992) 174:6184-6190,reports the transformation of various S. coelicolor mutants, lackingdifferent components of the act PKS gene cluster, with the correspondinggranaticin (gra) genes from S. violaceoruber, in trans.

[0012] However, no one to date has described the recombinant productionof polyketides using genetically engineered host cells whichsubstantially lack their entire native PKS gene clusters.

SUMMARY OF THE INVENTION

[0013] The present invention provides for novel polyketides and novelmethods of efficiently producing both new and known polyketides, usingrecombinant technology. In particular, a novel host-vector system isused to produce PKSs which in turn catalyze the production of a varietyof polyketides. Furthermore, methods are provided for the combinatorialbiosynthesis of polyketide libraries which can be screened for activecompounds. Such polyketides are useful as antibiotics, antitumor agents,immunosuppressants and for a wide variety of other pharmacologicalpurposes.

[0014] Accordingly, in one embodiment, the invention is directed to agenetically engineered cell which expresses a polyketide synthase (PKS)gene cluster in its native, nontransformed state, the geneticallyengineered cell substantially lacking the entire native PKS genecluster.

[0015] In another embodiment, the invention is directed to thegenetically engineered cell as described above, wherein the cellcomprises:

[0016] (a) a replacement PKS gene cluster which encodes a PKS capable ofcatalyzing the synthesis of a polyketide; and

[0017] (b) one or more control sequences operatively linked to the PKSgene cluster, whereby the genes in the gene cluster can be transcribedand translated in the genetically engineered cell,

[0018] with the proviso that when the replacement PKS gene clustercomprises an entire PKS gene set, at least one of the PKS genes orcontrol elements is heterologous to the cell.

[0019] In particularly preferred embodiments, the genetically engineeredcell is Streptomyces coelicolor, the cell substantially lacks the entirenative actinorhodin PKS gene cluster and the replacement PKS genecluster comprises a first gene encoding a PKS ketosynthase and a PKSacyltransferase active site (KS/AT), a second gene encoding a PKS chainlength determining factor (CLF), and a third gene encoding a PKS acylcarrier protein (ACP).

[0020] In another embodiment, the invention is directed to a method forproducing a recombinant polyketide comprising:

[0021] (a) providing a population of cells as described above; and

[0022] (b) culturing the population of cells under conditions wherebythe replacement PKS gene cluster present in the cells, is expressed.

[0023] In still another embodiment, the invention is directed to amethod for producing a—recombinant polyketide comprising:

[0024] (a) inserting a first portion of a replacement PKS gene clusterinto a donor plasmid and inserting a second portion of a replacement PKSgene cluster into a recipient plasmid, wherein the first and secondportions collectively encode a complete replacement PKS gene cluster,and further wherein:

[0025] i. the donor plasmid expresses a gene which encodes a firstselection marker and is capable of replication at a first, permissivetemperature and incapable of replication at a second, non-permissivetemperature;

[0026] ii. the recipient plasmid expresses a gene which encodes a secondselection marker; and

[0027] iii. the donor plasmid comprises regions of DNA complementary toregions of DNA in the recipient plasmid, such that homologousrecombination can occur between the first portion of the replacement PKSgene cluster and the second portion of the replacement gene cluster,whereby a complete replacement gene cluster can be generated;

[0028] (b) transforming the donor plasmid and the recipient plasmid intoa host cell and culturing the transformed host cell at the first,permissive temperature and under conditions which allow the growth ofhost cells which express the first and/or the second selection markers,to generate a first population of cells;

[0029] (c) culturing the first population of cells at the second,non-permissive temperature and under conditions which allow the growthof cells which express the first and/or the second selection markers, togenerate a second population of cells which includes host cells whichcontain a recombinant plasmid comprising a complete PKS replacement genecluster;

[0030] (d) transferring the recombinant plasmid from the secondpopulation of cells into the genetically engineered cell described aboveto generate transformed genetically engineered cells; and

[0031] (e) culturing the transformed genetically engineered cells underconditions whereby the replacement PKS gene cluster present in the cellsis expressed.

[0032] In a further embodiment, the invention is drawn to a method forpreparing a combinatorial polyketide library comprising:

[0033] (a) providing a population of vectors wherein the vectorscomprise a random assortment of polyketide synthase (PKS) genes,modules, active sites, or portions thereof and one or more controlsequences operatively linked to said genes;

[0034] (b) transforming a population of host cells with said populationof vectors;

[0035] (c) culturing said population of host cells under conditionswhereby the genes in said gene cluster can be transcribed andtranslated, thereby producing a combinatorial library of polyketides.

[0036] In still another embodiment, the invention is drawn to a methodfor producing a combinatorial polyketide library comprising:

[0037] a) providing one or more expression plasmids containing a randomassortment of 1 or more first modules of a modular PKS gene clusterwherein the expression plasmids express a gene which encodes a firstselection marker;

[0038] b) providing a pool of donor plasmids containing a randomassortment of second modules of a modular PKS gene cluster wherein thedonor plasmids express a gene which encodes a second selection markerand further wherein the donor plasmids comprise regions of DNAcomplementary to regions of DNA in the expression plasmids, such thathomologous recombination can occur between the first and second modules;

[0039] c) transforming the expression plasmids and the donor plasmidsinto a first population of host cells to produce a first pool oftransformed host cells;

[0040] d) culturing the first pool of transformed host cells underconditions which allow homologous recombination to occur between thefirst and second modules to produce recombined plasmids comprisingrecombined PKS gene cluster modules;

[0041] e) transferring the recombined plasmids into a second populationof host cells to generate a second pool of transformed host cells; and

[0042] f) culturing the second pool of transformed host cells underconditions whereby the combinatorial polyketide library is produced.

[0043] In yet another embodiment, the invention is directed to apolyketide compound having the structural formula (I)

[0044] wherein:

[0045] R¹ is selected from the group consisting of hydrogen and loweralkyl and R² is selected from the group consisting of hydrogen, loweralkyl and lower alkyl ester, or wherein R¹ and R² together form a loweralkylene bridge optionally substituted with one to four hydroxyl orlower alkyl groups;

[0046] R³ and R⁵ are independently selected from the group consisting ofhydrogen, halogen, lower alkyl, lower alkoxy, amino, lower alkyl mono-or di-substituted amino and nitro;

[0047] R⁴ is selected from the group consisting of halogen, lower alkyl,lower alkoxy, amino, lower alkyl mono- or di-substituted amino andnitro;

[0048] R⁶ is selected from the group consisting of hydrogen, loweralkyl, and —CHR⁷—(CO)R⁸ where R⁷ and R⁸ are independently selected fromthe group consisting of hydrogen and lower alkyl; and

[0049] i is 1, 2 or 3.

[0050] In another embodiment, the invention related to novel polyketideshaving the structures

[0051] In another embodiment, the invention is directed to a polyketidecompound formed by catalytic cyclization of an enzyme-bound ketidehaving the structure (II)

[0052] wherein:

[0053] R¹¹ is selected from the group consisting of methyl, —CH₂(CO)CH₃and —CH₂(CO)CH₂(CO)CH₃;

[0054] R¹² is selected from the group consisting of —S—E and—CH₂(CO)—S—E, wherein E represents a polyketide synthase produced by thegenetically engineered cells above; and

[0055] one of R¹³ and R¹⁴ is hydrogen and the other is hydroxyl, or R¹³and R¹⁴ together represent carbonyl.

[0056] In still another embodiment, the invention is directed to amethod for producing an aromatic polyketide, comprising effectingcyclization of an enzyme-bound ketide having the structure (II), whereincyclization is induced by the polyketide synthase.

[0057] In a further embodiment, the invention is directed to apolyketide compound having the structural formula (III)

[0058] wherein R² and R⁴ are as defined above and i is 0, 1 or 2.

[0059] In another embodiment, the invention is directed to a polyketidecompound having the structural formula (IV)

[0060] wherein R², R⁴ and i are as defined above for structural formula(III).

[0061] In still anther embodiment, the invention is directed to apolyketide compound having the structural formula (V)

[0062] wherein R², R⁴ and i are as defined above for structural formula(III).

[0063] These and other embodiments of the subject invention will readilyoccur to those of ordinary skill in the art in view of the disclosureherein.

BRIEF DESCRIPTION OF THE FIGURES

[0064]FIG. 1A shows the gene clusters for act, gra, and tcm PKSs andcyclases.

[0065]FIG. 1B shows the gene clusters for act, tcm, fren, gris, and whiEPKSs and cyclases.

[0066]FIG. 2 shows the strategy for making S. coelicolor CH999.

[0067]FIG. 2A depicts the structure of the act gene cluster present onthe S. coelicolor CH1 chromosome.

[0068]FIG. 2B shows the structure of pLRemEts and

[0069]FIG. 2C shows the portion of the CH999 chromosome with the actgene cluster deleted.

[0070]FIG. 3 is a diagram of plasmid pRM5.

[0071]FIG. 4 schematically illustrates formation of aloesaponarin II (2)and its carboxylated analog,3,8-dihydroxy-1-methylanthraquinone-2-carboxylic acid (1) as describedin Example 3.

[0072]FIG. 5 provides the structures of actinorhodin (3), granaticin(4), tetracenomycin (5) and mutactin (6), referenced in Example 4.

[0073]FIG. 6 schematically illustrates the preparation, via cyclizationof the polyketide precursors, of aloesaponarin II (2), its carboxylatedanalog, 3,8-dihydroxy-1-methylanthraquinone-2-carboxylic acid (1),tetracenomycin (5) and new compound RM20 (9), as explained in Example 4,part (A).

[0074]FIG. 7 schematically illustrates the preparation, via cyclizationof the polyketide precursors, of frenolicin (7), nanomycin (8) andactinorhodin (3).

[0075]FIG. 8 schematically illustrates the preparation, via cyclizationof the polyketide precursors, of novel compounds RM20 (9), RM18 (10),RM18b (11), SEK4 (12), SEK15 (13), RM20b (14), RM20c (15) and SEK15b(16).

[0076]FIG. 9 depicts the genetic model for the 6-deoxyerythronolide Bsynthase (DEBS).

[0077]FIG. 10 is a representation of the overall biosynthetic pathwayfor a typical polyketide natural product.

[0078]FIG. 11 shows the structures of various polyketide of aromatic,modular and fungal PKSs.

[0079]FIG. 12 is a scheme for rationally engineered biosynthesis ofpolyketides.

[0080]FIG. 13 shows the common moieties observed in engineeredpolyketides formed by non-enzymatic reactions involving the uncyclizedportions of the carbon chain. Hemiketals (a) and benzene rings (b) areformed at the methyl ends, whereas γ-pyrone rings (c) anddecarboxylations (d) occur at the carboxyl ends. The two chain ends canalso co-cyclize via aldol condensations (e).

[0081]FIG. 14 illustrates the structures and proposed pathways ofoctaketide-derived polyketides biosynthesis including RM77 (19).

[0082]FIG. 15 illustrates the structures and proposed pathways ofdecaketide-derived polyketides biosynthesis including RM80 (20) andRM80b (21).

[0083]FIG. 16 shows the structures of SEK34 (22) the two novelpolyketides SEK43 (23) and SEK26 (24) and other polyketides produced bygenetic engineering in S. coelicolor CH999.

[0084]FIG. 17 is a diagram of the proposed biosynthetic pathways for therationally designed polyketides SEK43 (23) and SEK26 (24).

[0085]FIG. 18 shows the strategy for the construction of recombinantmodular PKSs.

[0086]FIG. 19 is a diagram of plasmid pCK7.

[0087]FIG. 20 schematically illustrates the preparation of6-deoxyerythromolide B (17) from propionate and 8,8a-deoxyoleandolide(18) from an acetate starter.

[0088]FIG. 21A shows the biosynthesis of(2R,3S,4S,5R)-2,4-dimethyl-3,5-dihydroxy-n-heptanoic acid δ-lactone (25)by DEBS1 in S. coelicolor CH999. FIG. 21B shows the biosynthesis of (25)and (2R,3S,4S,5R)-2,4-dimethyl-3,5-dihydroxy-n-hexanoic acid δ-lactone(26) by the “1+2+TE” PKS in S. coelicolor CH999. The vertical linebetween ACP-2 and the TE represents the fusion junction in this deletionmutant.

[0089]FIG. 22 shows the biosynthesis of (8R,9s)-8,9-dihydro-8-methyl-9-hydroxy-10-deoxymethonolide (27) by the“1+2+3+4+5+TE” PKS in S. coelicolor CH999. The vertical line betweenKR-5 and ACP-6 represents the fusion junction in this deletion mutant.

DETAILED DESCRIPTION OF THE INVENTION

[0090] The practice of the present invention will employ, unlessotherwise indicated, conventional methods of chemistry, microbiology,molecular biology and recombinant DNA techniques within the skill of theart. Such techniques are explained fully in the literature. See, e.g.,Sambrook, et al. Molecular Cloning: A Laboratory Manual (CurrentEdition); DNA Cloning: A Practical Approach, vol. I & II (D. Glover,ed.); Oligonucleotide Synthesis (N. Gait, ed., Current Edition); NucleicAcid Hybridization (B. Hames & S. Higgins, eds., Current Edition);Transcription and Translation (B. Hames & S. Higgins, eds., CurrentEdition).

[0091] All publications, patents and patent applications cited herein,whether supra or infra, are hereby incorporated by reference in theirentirety.

[0092] As used in this specification and the appended claims, thesingular forms “a,” “an” and “the” include plural references unless thecontent clearly dictates otherwise. Thus, reference to “a polyketide”includes mixtures of polyketides, reference to “a polyketide synthase”includes mixtures of polyketide synthases, and the like.

[0093] A. Definitions

[0094] In describing the present invention, the following terms will beemployed, and are intended to be defined as indicated below.

[0095] By “replacement PKS gene cluster” is meant any set of PKS genescapable of producing a functional PKS when under the direction of one ormore compatible control elements, as defined below, in a host celltransformed therewith. A functional PKS is one which catalyzes thesynthesis of a polyketide. The term “replacement PKS gene cluster”encompasses one or more genes encoding for the various proteinsnecessary to catalyze the production of a polyketide. A “replacement PKSgene cluster” need not include all of the genes found in thecorresponding cluster in nature. Rather, the gene cluster need onlyencode the necessary PKS components to catalyze the production of anactive polyketide. Thus, as explained further below, if the gene clusterincludes, for example, eight genes in its native state and only three ofthese genes are necessary to provide an active polyketide, only thesethree genes need be present. Furthermore, the cluster can include PKSgenes derived from a single species, or may be hybrid in nature with,e.g., a gene derived from a cluster for the synthesis of a particularpolyketide replaced with a corresponding gene from a cluster for thesynthesis of another polyketide. Hybrid clusters can include genesderived from both Type I and Type II PKSs. As explained above, Type IPKSs include several large multifunctional proteins carrying, betweenthem, a set of separate active sites for each step of carbon chainassembly and modification. Type II PKSs, on the other hand, have asingle set of iteratively used active sites. These classifications arewell known. See, e.g., Hopwood, D. A. and Khosla, C. Secondarymetabolites: their function and evolution (1992) Wiley Chichester (CibaFoundation Symposium 171) p 88-112; Bibb, M. J. et al. EMBO J. (1989)8:2727; Sherman, D. H. et al. EMBO J. (1989) 8:2717; Fernandez-Moreno,M. A. et al. J. Biol. Chem. (1992) 267:19278); Cortes, J. et al. Nature(1990) 348:176; Donadio, S. et al. Science (1991) 252:675; MacNeil, D.J. et al. Gene (1992) 115:119. Hybrid clusters are exemplified hereinand are described further below. The genes included in the gene clusterneed not be the native genes, but can be mutants or analogs thereof.Mutants or analogs may be prepared by the deletion, insertion orsubstitution of one or more nucleotides of the coding sequence.Techniques for modifying nucleotide sequences, such as site-directedmutagenesis, are described in, e.g., Sambrook et al., supra; DNACloning, Vols. I and II, supra; Nucleic Acid Hybridization, supra.

[0096] A “replacement PKS gene cluster” may also contain genes codingfor modifications to the core polyketide catalyzed by the PKS,including, for example, genes encoding post-polyketide synthesis enzymesderived from natural products pathways such as O-methyl-transferases andglycosyltransferases. A “replacement PKS gene cluster” may furtherinclude genes encoding hydroxylases, methylases or other alkylases,oxidases, reductases, glycotransferases, lyases, ester or amidesynthases, and various hydrolases such as esterases and amidases.

[0097] As explained further below, the genes included in the replacementgene cluster need not be on the same plasmid or if present on the sameplasmid, can be controlled by the same or different control sequences.

[0098] A “library” or “combinatorial library” of polyketides is intendedto mean a collection of polyketides catalytically produced by a PKS genecluster capable of catalyzing the synthesis of a polyketide. The librarycan be produced by a PKS gene cluster that contains any combination ofnative, homolog or mutant genes from aromatic, modular or fungal PKSs.The combination of genes can be derived from a single PKS gene cluster,e.g., act, fren, gra, tcm, whiE, gris, ery, or the like, and mayoptionally include genes encoding tailoring enzymes which are capable ofcatalyzing the further modification of a polyketide. Alternatively, thecombination of genes can be rationally or stochastically derived from anassortment of PKS gene clusters, e.g. a minimal PKS gene cluster can beconstructed to contain the KS/AT component from an act PKS, the CLFcomponent from a tcm PKS and a ACP component from a fren PKS. Thecombination of genes can optionally include KR, CYC and ARO componentsof PKS gene clusters as well. The library of polyketides thus producedcan be tested or screened for biological, pharmacological or otheractivity.

[0099] By “random assortment” is intended any combination and/or orderof genes, homologs or mutants which encode for the various PKS enzymes,modules, active sites or portions thereof derived from aromatic, modularor fungal PKS gene clusters.

[0100] By “genetically engineered host cell” is meant a host cell wherethe native PKS gene cluster has been deleted using recombinant DNAtechniques or host cell into which a heterologous PKS gene cluster hasbeen inserted. Thus, the term would not encompass mutational eventsoccurring in nature. A “host cell” is a cell derived from a procaryoticmicroorganism or a eucaryotic cell line cultured as a unicellularentity, which can be, or has been, used as a recipient for recombinantvectors bearing the PKS gene clusters of the invention. The termincludes the progeny of the original cell which has been transfected. Itis understood that the progeny of a single parental cell may notnecessarily be completely identical in morphology or in genomic or totalDNA complement to the original parent, due to accidental or deliberatemutation. Progeny of the parental cell which are sufficiently similar tothe parent to be characterized by the relevant property, such as thepresence of a nucleotide sequence encoding a desired PKS, are includedin the definition, and are covered by the above terms.

[0101] The term “heterologous” as it relates to nucleic acid sequencessuch as coding sequences and control sequences, denotes sequences thatare not normally associated with a region of a recombinant construct,and/or are not normally associated with a particular cell. Thus, a“heterologous” region of a nucleic acid construct is an identifiablesegment of nucleic acid within or attached to another nucleic acidmolecule that is not found in association with the other molecule innature. For example, a heterologous region of a construct could includea coding sequence flanked by sequences not found in association with thecoding sequence in nature. Another example of a heterologous codingsequence is a construct where the coding sequence itself is not found innature (e.g., synthetic sequences having codons different from thenative gene). Similarly, a host cell transformed with a construct whichis not normally present in the host cell would be consideredheterologous for purposes of this invention. Allelic variation ornaturally occurring mutational events do not give rise to heterologousDNA, as used herein.

[0102] A “coding sequence” or a sequence which “encodes” a particularPKS, is a nucleic acid sequence which is transcribed (in the case ofDNA) and translated (in the case of mRNA) into a polypeptide in vitro orin vivo when placed under the control of appropriate regulatorysequences. The boundaries of the coding sequence are determined by astart codon at the 5′ (amino) terminus and a translation stop codon atthe 3′ (carboxy) terminus. A coding sequence can include, but is notlimited to, cDNA from procaryotic or eucaryotic mRNA, genomic DNAsequences from procaryotic or eucaryotic DNA, and even synthetic DNAsequences. A transcription termination sequence will usually be located3′ to the coding sequence.

[0103] A “nucleic acid” sequence can include, but is not limited to,procaryotic sequences, eucaryotic mRNA, cDNA from eucaryotic mRNA,genomic DNA sequences from eucaryotic (e.g., mammalian) DNA, and evensynthetic DNA sequences. The term also captures sequences that includeany of the known base analogs of DNA and RNA such as, but not limited to4-acetylcytosine, 8-hydroxy-N6-methyladenosine, aziridinylcytosine,pseudoisocytosine, 5-(carboxyhydroxylmethyl) uracil, 5-fluorouracil,5-bromouracil, 5-carboxymethylaminomethyl-2-thiouracil,5-carboxymethylaminomethyluracil, dihydrouracil, inosine,N6-isopentenyladenine, 1-methyladenine, 1-methylpseudouracil,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-methyladenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarbonylmethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid methylester,uracil-5-oxyacetic acid, oxybutoxosine, pseudouracil, queosine,2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil,5-methyluracil, N-uracil-5-oxyacetic acid methylester,uracil-5-oxyacetic acid, pseudouracil, queosine, 2-thiocytosine, and2,6-diaminopurine. A transcription termination sequence will usually belocated 3′ to the coding sequence.

[0104] DNA “control sequences” refers collectively to promotersequences, ribosome binding sites, polyadenylation signals,transcription termination sequences, upstream regulatory domains,enhancers, and the like, which collectively provide for thetranscription and translation of a coding sequence in a host cell. Notall of these control sequences need always be present in a recombinantvector so long as the desired gene is capable of being transcribed andtranslated.

[0105] “Operably linked” refers to an arrangement of elements whereinthe components so described are configured so as to perform their usualfunction. Thus, control sequences operably linked to a coding sequenceare capable of effecting the expression of the coding sequence. Thecontrol sequences need not be contiguous with the coding sequence, solong as they function to direct the expression thereof. Thus, forexample, intervening untranslated yet transcribed sequences can bepresent between a promoter sequence and the coding sequence and thepromoter sequence can still be considered “operably linked” to thecoding sequence.

[0106] By “selection marker” is meant any genetic marker which can beused to select a population of cells which carry the marker in theirgenome. Examples of selection markers include: auxotrophic markers bywhich cells are selected by their ability to grow on minimal media withor without a nutrient or supplement, e.g., thymidine, diaminopimelicacid or biotin; metabolic markers by which cells are selected for theirability to grow on minimal media containing the appropriate sugar as thesole carbon source or the ability of cells to form colored coloniescontaining the appropriate dyes or chromogenic substrates; and drugresistance markers by which cells are selected by their ability to growon media containing one or more of the appropriate drugs, e.g.,tetracycline, ampicillin, kanamycin, streptomycin or nalidixic acid.

[0107] “Recombination” is a the reassortment of sections of DNAsequences between two DNA molecules. “Homologous recombination” occursbetween two DNA molecules which hybridize by virtue of homologous orcomplementary nucleotide sequences present in each DNA molecule.

[0108] The term “alkyl” as used herein refers to a branched orunbranched saturated hydrocarbon group of 1 to 24 carbon atoms, such asmethyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, octyl,decyl, tetradecyl, hexadecyl, eicosyl, tetracosyl and the like.Preferred alkyl groups herein contain 1 to 12 carbon atoms. The term“lower alkyl” intends an alkyl group of one to six carbon atoms,preferably one to four carbon atoms.

[0109] The term “alkylene” as used herein refers to a difunctionalsaturated branched or unbranched hydrocarbon chain containing from 1 to24 carbon atoms, and includes, for example, methylene (—CH₂—), ethylene(—CH₂—CH₂—), propylene (—CH₂—CH₂—CH₂—), 2-methylpropylene[—CH₂—CH(CH₃)—CH₂—], hexylene [—(CH₂)₆—] and the like. “Lower alkylene”refers to an alkylene group of 1 to 6, more preferably 1 to 4, carbonatoms.

[0110] The term “alkoxy” as used herein intends an alkyl group boundthrough a single, terminal ether linkage; that is, an “alkoxy” group maybe defined as —OR where R is alkyl as defined above. A “lower alkoxy”group intends an alkoxy group containing one to six, more preferably oneto four, carbon atoms.

[0111] “Halo” or “halogen” refers to fluoro, chloro, bromo or iodo, andusually relates to halo substitution for a hydrogen atom in an organiccompound. Of the halos, chloro and fluoro are generally preferred.

[0112] “Optional” or “optionally” means that the subsequently describedevent or circumstance may or may not occur, and that the descriptionincludes instances where said event or circumstance occurs and instanceswhere it does not. For example, the phrase “optionally substitutedalkylene” means that an alkylene moiety may or may not be substitutedand that the description includes both unsubstituted alkylene andalkylene where there is substitution.

[0113] B. General Methods

[0114] Central to the present invention is the discovery of ahost-vector system for the efficient recombinant production of bothnovel and known polyketides. In particular, the invention makes use ofgenetically engineered cells which have their naturally occurring PKSgenes substantially deleted. These host cells can be transformed withrecombinant vectors, encoding a variety of PKS gene clusters, for theproduction of active polyketides. The invention provides for theproduction of significant quantities of product at an appropriate stageof the growth cycle. The polyketides so produced can be used astherapeutic agents, to treat a number of disorders, depending on thetype of polyketide in question. For example, several of the polyketidesproduced by the present method will find use as immunosuppressants, asanti-tumor agents, as well as for the treatment of viral, bacterial andparasitic infections. The ability to recombinantly produce polyketidesalso provides a powerful tool for characterizing PKSs and the mechanismof their actions.

[0115] More particularly, host cells for the recombinant production ofthe subject polyketides can be derived from any organism with thecapability of harboring a recombinant PKS gene cluster. Thus, the hostcells of the present invention can be derived from either procaryotic oreucaryotic organisms. However, preferred host cells are thoseconstructed from the actinomycetes, a class of mycelial bacteria whichare abundant producers of a number of polyketides. A particularlypreferred genus for use with the present system is Streptomyces. Thus,for example, S. ambofaciens, S. avermitilis, S. azureus, S.cinnamonensis, S. coelicolor, S. curacoi, S. erythraeus, S. fradiae, S.galilaeus, S. glaucescens, S. hygroscopicus, S. lividans, S. parvulus,S. peucetius, S. rimosus, S. roseofulvus, S. thermotolerans, S.violaceoruber, among others, will provide convenient host cells for thesubject invention, with S. coelicolor being preferred. (See, e.g.,Hopwood, D. A. and Sherman, D. H. Ann. Rev. Genet. (1990) 24:37-66;O'Hagan, D. The Polyketide Metabolites (Ellis Horwood Limited, 1991),for a description of various polyketide-producing organisms and theirnatural products.)

[0116] The above-described cells are genetically engineered by deletingthe naturally occurring PKS genes therefrom, using standard techniques,such as by homologous recombination. (See, e.g., Khosla, C. et al.Molec. Microbiol. (1992) 6:3237). Exemplified herein is a geneticallyengineered S. coelicolor host cell. Native strains of S. coelicolorproduce a PKS which catalyzes the biosynthesis of the aromaticpolyketide actinorhodin (structure 3, FIG. 5). The novel strain, S.coelicolor CH999 (as described in the examples), was constructed bydeleting, via homologous recombination, the entire natural act clusterfrom the chromosome of S. coelicolor CH1 (FIG. 2) (Khosla, C. Molec.Microbiol. (1992) 6:3237), a strain lacking endogenous plasmids andcarrying a stable mutation that blocks biosynthesis of another pigmentedS. coelicolor antibiotic, undecylprodigiosin.

[0117] The host cells described above can be transformed with one ormore vectors, collectively encoding a functional PKS set, or a cocktailcomprising a random assortment of PKS genes, modules, active sites, orportions thereof. The vector(s) can include native or hybridcombinations of PKS subunits or cocktail components, or mutants thereof.As explained above, the replacement gene cluster need not correspond tothe complete native gene cluster but need only encode the necessary PKScomponents to catalyze the production of a polyketide. For example, ineach Streptomyces aromatic PKS so far studied, carbon chain assemblyrequires the products of three open reading frames (ORFs). ORF1 encodesa ketosynthase (KS) and an acyltransferase (AT) active site (KS/AT); aselucidated herein, ORF2 encodes a chain length determining factor (CLF),a protein similar to the ORF1 product but lacking the KS and AT motifs;and ORF3 encodes a discrete acyl carrier protein (ACP). Some geneclusters also code for a ketoreductase (KR) and a cyclase, involved incyclization of the nascent polyketide backbone. (See FIGS. 1A and 1B forschematic representations of six PKS gene clusters.) However, it hasbeen found that only the KS/AT, CLF, and ACP, need be present in orderto produce an identifiable polyketide. Thus, in the case of aromaticPKSs derived from Streptomyces, these three genes, without the othercomponents of the native clusters, can be included in one or morerecombinant vectors, to constitute a “minimal” replacement PKS genecluster.

[0118] Furthermore, the recombinant vector(s) can include genes from asingle PKS gene cluster, or may comprise hybrid replacement PKS geneclusters with, e.g., a gene for one cluster replaced by thecorresponding gene from another gene cluster. For example, it has beenfound that ACPs are readily interchangeable among different synthaseswithout an effect on product structure. Furthermore, a given KR canrecognize and reduce polyketide chains of different chain lengths.Accordingly, these genes are freely interchangeable in the constructsdescribed herein. Thus, the replacement clusters of the presentinvention can be derived from any combination of PKS gene sets whichultimately function to produce an identifiable polyketide.

[0119] Examples of hybrid replacement clusters include clusters withgenes derived from two or more of the act gene cluster, the whiE genecluster, frenolicin (fren), granaticin (gra), tetracenomycin (tcm),6-methylsalicylic acid (6-msas), oxytetracycline (otc), tetracycline(tet), erythromycin (ery), griseusin (gris), nanaomycin, medermycin,daunorubicin, tylosin, carbomycin, spiramycin, avermectin, monensin,nonactin, curamycin, rifamycin and candicidin synthase gene clusters,among others. (For a discussion of various PKSs, see, e.g., Hopwood, D.A. and Sherman, D. H. Ann. Rev. Genet. (1990) 24:37-66; O'Hagan, D. ThePolyketide Metabolites (Ellis Horwood Limited, 1991).)

[0120] More particularly, a number of hybrid gene clusters have beenconstructed herein, having components derived from the act, fren, tcm,gris and gra gene clusters, as depicted in Tables 1, 2, 5 and 6. Severalof the hybrid clusters were able to functionally express both novel andknown polyketides in S. coelicolor CH999 (described above). However,other hybrid gene clusters, as described above, can easily be producedand screened using the disclosure herein, for the production ofidentifiable polyketides.

[0121] Furthermore, a library of randomly cloned ORF1, ORF2, ORF3 andhomologs or mutant thereof, as well as other PKS genes and homologs ormutants thereof including ketoreductases, cyclases and aromatases from acollection of aromatic PKS gene clusters, could be constructed andscreened for identifiable polyketides using methods described andexemplified herein. In addition, a considerable degree of variabilityexists for both the starter units (e.g., acetyl CoA, maloamyl CoA,propionyl CoA, acetate, butyrate, isobutyrate and the like) and theextender units among certain naturally occurring aromatic PKSs; thus,these units can also be used for obtaining novel polyketides via geneticengineering.

[0122] Additionally, a library of randomly cloned open reading frames orhomologs from a collection of modular PKS gene clusters could beconstructed and screened for identifiable polyketides. Such geneclusters are described in further detail below. Recombinant vectors canoptionally include genes from an aromatic and a modular PKS genecluster.

[0123] The recombinant vectors, harboring the gene clusters or randomassortment of PKS genes, modules, active sites or portions thereofdescribed above, can be conveniently generated using techniques known inthe art. For example, the PKS subunits of interest can be obtained froman organism that expresses the same, using recombinant methods, such asby screening cDNA or genomic libraries, derived from cells expressingthe gene, or by deriving the gene from a vector known to include thesame. The gene can then be isolated and combined with other desired PKSsubunits, using standard techniques. If the gene in question is alreadypresent in a suitable expression vector, it can be combined in situ,with, e.g., other PKS subunits, as desired. The gene of interest canalso be produced synthetically, rather than cloned. The nucleotidesequence can be designed with the appropriate codons for the particularamino acid sequence desired. In general, one will select preferredcodons for the intended host in which the sequence will be expressed.The complete sequence can be assembled from overlapping oligonucleotidesprepared by standard methods and assembled into a complete codingsequence. See, e.g., Edge (1981) Nature 292:756; Nambair et al. (1984)Science 223:1299; Jay et al. (1984) J. Biol. Chem. 259:6311.

[0124] Mutations can be made to the native PKS subunit sequences andsuch mutants used in place of the native sequence, so long as themutants are able to function with other PKS subunits to collectivelycatalyze the synthesis of an identifiable polyketide. Such mutations canbe made to the native sequences using conventional techniques such as bypreparing synthetic oligonucleotides including the mutations andinserting the mutated sequence into the gene encoding a PKS subunitusing restriction endonuclease digestion. (See, e.g., Kunkel, T. A.Proc. Natl. Acad. Sci. USA (1985) 82:448; Geisselsoder et al.BioTechniques (1987) 5:786.) Alternatively, the mutations can beeffected using a mismatched primer (generally 10-20 nucleotides inlength) which hybridizes to the native nucleotide sequence (generallycDNA corresponding to the RNA sequence), at a temperature below themelting temperature of the mismatched duplex. The primer can be madespecific by keeping primer length and base composition within relativelynarrow limits and by keeping the mutant base centrally located. Zollerand Smith, Methods Enzymol. (1983) 100:468. Primer extension is effectedusing DNA polymerase, the product cloned and clones containing themutated DNA, derived by segregation of the primer extended strand,selected. Selection can be accomplished using the mutant primer as ahybridization probe. The technique is also applicable for generatingmultiple point mutations. See, e.g., Dalbie-McFarland et al. Proc. Natl.Acad. Sci USA (1982) 79:6409. PCR mutagenesis will also find use foreffecting the desired mutations.

[0125] Random mutagenesis of the nucleotide sequences obtained asdescribed above can be accomplished by several different techniquesknown in the art, such as by altering sequences within restrictionendonuclease sites, inserting an oligonucleotide linker randomly into aplasmid, by irradiation with X-rays or ultraviolet light, byincorporating incorrect nucleotides during in vitro DNA synthesis, byerror-prone PCR mutagenesis, by preparing synthetic mutants or bydamaging plasmid DNA in vitro with chemicals. Chemical mutagens include,for example, sodium bisulfite, nitrous acid, hydroxylamine, agents whichdamage or remove bases thereby preventing normal base-pairing such ashydrazine or formic acid, analogues of nucleotide precursors such asnitrosoguanidine, 5-bromouracil, 2-aminopurine, or acridineintercalating agents such as proflavine, acriflavine, quinacrine, andthe like. Generally, plasmid DNA or DNA fragments are treated withchemicals, transformed into E. coli and propagated as a pool or libraryof mutant plasmids.

[0126] Large populations of random enzyme variants can be constructed invivo using “recombination-enhanced mutagenesis.” This method employs twoor more pools of, for example, 10⁶ mutants each of the wild-typeencoding nucleotide sequence that are generated using any convenientmutagenesis technique, described more fully above, and then insertedinto cloning vectors.

[0127] Once the mutant sequences are generated, the DNA is inserted intoan appropriate cloning vector, using techniques well known in the art(see, e.g., Sambrook et al., supra). The choice of vector depends on thepool of mutant sequences, i.e., donor or recipient, with which they areto be employed. Furthermore, the choice of vector determines the hostcell to be employed in subsequent steps of the claimed method. Anytransducible cloning vector can be used as a cloning vector for thedonor pool of mutants. It is preferred, however, that phagemids,cosmids, or similar cloning vectors be used for cloning the donor poolof mutant encoding nucleotide sequences into the host cell. Phagemidsand cosmids, for example, are advantageous vectors due to the ability toinsert and stably propagate therein larger fragments of DNA than in M13phage and λ phage, respectively. Phagemids which will find use in thismethod generally include hybrids between plasmids and filamentous phagecloning vehicles. Cosmids which will find use in this method generallyinclude λ phage-based vectors into which cos sites have been inserted.Recipient pool cloning vectors can be any suitable plasmid. The cloningvectors into which pools of mutants are inserted may be identical or maybe constructed to harbor and express different genetic markers (see,e.g., Sambrook et al., supra). The utility of employing such vectorshaving different marker genes may be exploited to facilitate adetermination of successful transduction.

[0128] Thus, for example, the cloning vector employed may be a phagemidand the host cell may be E. coli. Upon infection of the host cell whichcontains a phagemid, single-stranded phagemid DNA is produced, packagedand extruded from the cell in the form of a transducing phage in amanner similar to other phage vectors. Thus, clonal amplification ofmutant encoding nucleotide sequences carried by phagemids isaccomplished by propagating the phagemids in a suitable host cell.

[0129] Following clonal amplification, the cloned donor pool of mutantsis infected with a helper phage to obtain a mixture of phage particlescontaining either the helper phage genome or phagemids mutant alleles ofthe wild-type encoding nucleotide sequence.

[0130] Infection, or transfection, of host cells with helper phage isgenerally accomplished by methods well known in the art (see, e.g.,Sambrook et al., supra; and Russell et al. (1986) Gene 45:333-338).

[0131] The helper phage may be any phage which can be used incombination with the cloning phage to produce an infective transducingphage. For example, if the cloning vector is a cosmid, the helper phagewill necessarily be a λ phage. Preferably, the cloning vector is aphagemid and the helper phage is a filamentous phage, and preferablyphage M13.

[0132] If desired after infecting the phagemid with helper phage andobtaining a mixture of phage particles, the transducing phage can beseparated from helper phage based on size differences (Barnes et al.(1983) Methods Enzymol. 101:98-122), or other similarly effectivetechnique.

[0133] The entire spectrum of cloned donor mutations can now betransduced into clonally amplified recipient cells into which has beentransduced or transformed a pool of mutant encoding nucleotidesequences. Recipient cells which may be employed in the method disclosedand claimed herein may be, for example, E. coli, or other bacterialexpression systems which are not recombination deficient. Arecombination deficient cell is a cell in which recombinatorial eventsis greatly reduced, such as the rec⁻ mutants of E. coli (see, Clark etal. (1965) Proc. Natl. Acad. Sci. USA 53:451-459).

[0134] By maintaining a high multiplicity of infection (MOI) and a ratioof [transductant forming units (tfu)]:[plaque forming units (pfu)]greater than 1, one can insure that virtually every recipient cellreceives at least one mutant gene from the donor pool. The MOI isadjusted by manipulating the ratio of transducing particles to celldensity. By the term “high multiplicity of infection” is meant amultiplicity of infection of greater than 1, preferably between 1 to100, more preferably between 1 and 10.

[0135] It is preferred that the tfu:pfu ratio, as reflecting the ratioof transducing phages to helper phages, be as large as possible, atleast greater than one, more preferably greater than 100 or more. Byexercising the option to separate transducing phage from helper phage,as described above, the tfu:pfu ratio can be maximized.

[0136] These transductants can now be selected for the desired expressedprotein property or characteristic and, if necessary or desirable,amplified. Optionally, if the phagemids into which each pool of mutantsis cloned are constructed to express different genetic markers, asdescribed above, transductants may be selected by way of theirexpression of both donor and recipient plasmid markers.

[0137] The recombinants generated by the above-described methods canthen be subjected to selection or screening by any appropriate method,for example, enzymatic or other biological activity.

[0138] The above cycle of amplification, infection, transduction, andrecombination may be repeated any number of times using additional donorpools cloned on phagemids. As above, the phagemids into which each poolof mutants is cloned may be constructed to express a different markergene. Each cycle could increase the number of distinct mutants by up toa factor of 10⁶. Thus, if the probability of occurrence of aninter-allelic recombination event in any individual cell is f (aparameter that is actually a function of the distance between therecombining mutations), the transduced culture from two pools of 10⁶allelic mutants will express up to 10¹² distinct mutants in a populationof 10¹²/f cells.

[0139] The gene sequences, native or mutant, which collectively encode areplacement PKS gene cluster, can be inserted into one or moreexpression vectors, using methods known to those of skill in the art. Inorder to incorporate a random assortment of PKS genes, modules, activesites or portions thereof into am expression vector, a cocktail of samecan be prepared and used to generate the expression vector by techniqueswell known in the art and described in detail below. Expression vectorswill include control sequences operably linked to the desired PKS codingsequence. Suitable expression systems for use with the present inventioninclude systems which function in eucaryotic and procaryotic host cells.However, as explained above, procaryotic systems are preferred, and inparticular, systems compatible with Streptomyces spp. are of particularinterest. Control elements for use in such systems include promoters,optionally containing operator sequences, and ribosome binding sites.Particularly useful promoters include control sequences derived from PKSgene clusters which result in the production of polyketides as secondarymetabolites, such as one or more act promoters, tcm promoters,spiramycin promoters, and the like. However, other bacterial promoters,such as those derived from sugar metabolizing enzymes, such asgalactose, lactose (lac) and maltose, will also find use in the presentconstructs. Additional examples include promoter sequences derived frombiosynthetic enzymes such as tryptophan (trp), the β-lactamase (bla)promoter system, bacteriophage lambda PL, and T5. In addition, syntheticpromoters, such as the tac promoter (U.S. Pat. No. 4,551,433), which donot occur in nature also function in bacterial host cells.

[0140] Other regulatory sequences may also be desirable which allow forregulation of expression of the PKS replacement sequences relative tothe growth of the host cell. Regulatory sequences are known to those ofskill in the art, and examples include those which cause the expressionof a gene to be turned on or off in response to a chemical or physicalstimulus, including the presence of a regulatory compound. Other typesof regulatory elements may also be present in the vector, for example,enhancer sequences.

[0141] Selectable markers can also be included in the recombinantexpression vectors. A variety of markers are known which are useful inselecting for transformed cell lines and generally comprise a gene whoseexpression confers a selectable phenotype on transformed cells when thecells are grown in an appropriate selective medium. Such markersinclude, for example, genes which confer antibiotic resistance orsensitivity to the plasmid. Alternatively, several polyketides arenaturally colored and this characteristic provides a built-in marker forselecting cells successfully transformed by the present constructs.

[0142] The various PKS subunits of interest, or the cocktail of PKSgenes, modules, active sites, or portions thereof, can be cloned intoone or more recombinant vectors as individual cassettes, with separatecontrol elements, or under the control of, e.g., a single promoter. ThePKS subunits or cocktail components can include flanking restrictionsites to allow for the easy deletion and insertion of other PKS subunitsor cocktail components so that hybrid PKSs can be generated. The designof such unique restriction sites is known to those of skill in the artand can be accomplished using the techniques described above, such assite-directed mutagenesis and PCR.

[0143] Using these techniques, a novel plasmid, pRM5, (FIG. 3 andExample 2) was constructed as a shuttle vector for the production of thepolyketides described herein. Plasmid pRM5 includes the act genesencoding the KS/AT (ORF1), CLF (ORF2) and ACP (ORF3) PKS subunits,flanked by PacI, NsiI and XbaI restriction sites. Thus, analogous PKSsubunits, encoded by other PKS genes, can be easily substituted for theexisting act genes. (See, e.g., Example 4, describing the constructionof hybrid vectors using pRM5 as the parent plasmid). The shuttle plasmidalso contains the act KR gene (actIII), the cyclase gene (actVII), and aputative dehydratase gene (actIV), as well as a ColEI replicon (to allowtransformation of E. coli), an appropriately truncated SCP2* (low copynumber) Streptomyces replicon, and the actII-ORF4 activator gene fromthe act cluster, which induces transcription from act promoters duringthe transition from growth phase to stationary phase in the vegetativemycelium. pRM5 carries the divergent actI/actIII promoter pair.

[0144] Methods for introducing the recombinant vectors of the presentinvention into suitable hosts are known to those of skill in the art andtypically include the use of CaCl₂ or other agents, such as divalentcations and DMSO. DNA can also be introduced into bacterial cells byelectroporation. Once the PKSs are expressed, the polyketide producingcolonies can be identified and isolated using known techniques. Theproduced polyketides can then be further characterized.

[0145] As explained above, the above-described recombinant methods alsofind utility in the catalytic biosynthesis of polyketides by large,modular PKSs. For example, 6-deoxyerythronolide B synthase (DEBS)catalyzes the biosynthesis of the erythromycin aglycone,6-deoxyerythronolide B (17). Three open reading frames (eryAI, eryAII,and eryAIII) encode the DEBS polypeptides and span 32 kb in the ery genecluster of the Saccharopolyspora erythraea genome. The genes areorganized in six repeated units, each designated a “module.” Each moduleencodes a set of active sites that, during polyketide biosynthesis,catalyzes the condensation of an additional monomer onto the growingchain. Each module includes an acyltransferase (AT), β-ketoacyl carrierprotein synthase (KS), and acyl carrier protein (ACP) as well as asubset of reductive active sites (β-ketoreductase (KR), dehydratase(DH), enoyl reductase (ER)) (FIG. 9). The number of reductive siteswithin a module corresponds to the extent of β-keto reduction in eachcondensation cycle. The thioesterase (TE) encoded at the end of moduleappears to catalyze lactone formation.

[0146] Due to the large sizes of eryAI, eryAII, and eryAIII, and thepresence of multiple active sites, these genes can be convenientlycloned into a plasmid suitable for expression in a geneticallyengineered host cell, such as CH999, using an in vivo recombinationtechnique. This technique, described in Example 7 and summarized in FIG.10, utilizes derivatives of the plasmid pMAK705 (Hamilton et al. (1989)J. Bacteriol. 171:4617) to permit in vivo recombination between atemperature-sensitive donor plasmid, which is capable of replication ata first, permissive temperature and incapable of replication at asecond, non-permissive temperature, and recipient plasmid. The eryAgenes thus cloned gave pCK7, a derivative of pRM5 (McDaniel et al.(1993) Science 262:1546). A control plasmid, pCK7f, was constructed tocarry a frameshift mutation in eryAI. pCK7 and pCK7f possess a ColEIreplicon for genetic manipulation in E. coli as well as a truncatedSCP2* (low copy number) Streptomyces replicon. These plasmids alsocontain the divergent actI/actIII promoter pair and actII-ORF4, anactivator gene, which is required for transcription from these promotersand activates expression during the transition from growth to stationaryphase in the vegetative mycelium. High-level expression of PKS genesoccurs at the onset of stationary phase of mycelial growth; therecombinant strains therefore produce “reporter” polyketides assecondary metabolites in a quasi-natural manner.

[0147] Recombinant vectors harboring modular PKSs can also be generatedusing techniques known in the art. For example, the PKS of interest canbe obtained from an organism that expresses the same using recombinanttechniques as describe above and exemplified in Examples 7 and 8. Forexample, the gene can be isolated, subjected to mutation-producingprotocols and reexpressed (see Example 8).

[0148] The method described above for producing polyketides synthesizedby large, modular PKSs may be used to produce other polyketides assecondary, metabolites such as sugars, β-lactams, fatty acids,aminoglycosides, terpinoids, non-ribosomal peptides, prostanoid hormonesand the like. In this manner, the polyketides can be produced after thehost cell has matured, thereby reducing any potential toxic or otherbioactive effects of the polyketide on the host cell.

[0149] As with aromatic (Type II) and modular (Type I) PKSs, the abovedescribed methods also find utility in the catalytic biosynthesis ofpolyketides using the PKS genes from fungi. Fungal PKSs, such as the6-methylsalicylic acid PKS consist of a single multi-domain polypeptidewhich includes all active sites required for the biosynthesis of6-methylsalicylic acid.

[0150] Using the above recombinant methods, a number of polyketides havebeen produced. These compounds have the general structure (I)

[0151] wherein R¹, R², R³, R⁴, R⁵, R⁶ R⁷, R⁸ and i are as defined above.One group of such compounds are wherein: R¹ is lower alkyl, preferablymethyl; R², R³ and R⁶ are hydrogen; R⁶ is —CHR⁷—(CO)—R⁸; and i is 0. Asecond group of such compounds are wherein: R¹ and R⁶ are lower alkyl,preferably methyl; R², R³ and R⁵ are hydrogen; and i is 0. Still a thirdgroup of such compounds are wherein: R¹ and R² are linked together toform a lower alkylene bridge —CHR⁹—CHR¹⁰ wherein R⁹ and R¹⁰ areindependently selected from the group consisting of hydrogen, hydroxyland lower alkyl, e.g., —CH₂—CHOH—; R³ and R⁵ are hydrogen; R⁶ is—CHR⁷—(CO)—R⁸ where R⁸ is hydrogen or lower alkyl, e.g., —CH₂—(CO)—CH₃;and i is 0. Specific such compounds include the following compounds 9,10 and 11 as follows:

[0152] Other novel polyketides within the scope of the invention arethose having the structure

[0153] Preparation of compounds 9, 10, 11, 12, 13, 14, 15 and 16 iseffected by cyclization of an enzyme-bound polyketide having thestructure (II)

[0154] wherein R¹¹, R¹², R¹³ and R¹⁴ and E are as defined earlierherein. Examples of such compounds include: a first group wherein R¹¹ ismethyl and R¹² is —CH₂(CO)—S—E; a second group wherein R¹¹ is—CH₂(CO)CH₃ and R¹² is —S—E; a third group wherein R¹¹ is —CH₂(CO)CH₃and R¹² is —CH₂(CO)—S—E; and a fourth group wherein R¹¹ is—CH₂(CO)CH₂(CO)CH₃ and R¹² is —CH₂(CO)—S—E (see FIG. 8 for structuralexemplification).

[0155] The remaining structures encompassed by generic formula (I)—i.e.,structures other than 9, 10 and 11—may be prepared from structures 9, 10or 11 using routine synthetic organic methods well-known to thoseskilled in the art of organic chemistry, e.g., as described by H. O.House, Modern Synthetic Reactions, Second Edition (Menlo Park, Calif.:The Benjamin/Cummings Publishing Company, 1972), or by J. March,Advanced Organic Chemistry: Reactions, Mechanisms and Structure, 4th Ed.(New York: Wiley-Interscience, 1992), the disclosures of which arehereby incorporated by reference. Typically, as will be appreciated bythose skilled in the art, incorporation of substituents on the aromaticrings will involve simple electrophilic aromatic addition reactions.Structures 12 and 13 may be modified in a similar manner to producepolyketides which are also intended to be within the scope of thepresent invention.

[0156] In addition, the above recombinant methods have been used toproduce polyketide compound having the general structure (III)

[0157] general structure (IV)

[0158] and general structure (V)

[0159] Particularly preferred compounds of structural formulas (III),(IV) and (V) are wherein: R² is hydrogen and i is 0.

[0160] As disclosed hereinabove and in the Examples which follow, asystem has been developed to functionally express recombinant PKSs andto produce novel aromatic polyketides (Examples 1-6). This technologyhas been extrapolated to larger gene clusters using an in vivorecombination strategy (Kao et al. Science (1994) 265:509-512; seeExamples 7 and 8). These systems may be used to genetically manipulatepolyketide biosynthesis to generate libraries of synthetic products.

[0161] A typical pathway for polyketide biosynthesis is shown in FIG.10. Generally, polyketide synthesis occurs in three stages. In the firststage, catalyzed by the PKS, a nascent polyketide backbone is generatedfrom monomeric CoA thioesters. In the second stage this backbone isregiospecifically cyclized. While some cyclization reactions arecontrolled by the PKS itself, others result from activities ofdownstream enzymes. In the final stage, the cyclized intermediate ismodified further by the action of mechanistically diverse “tailoringenzymes,” giving rise to the natural product.

[0162] More particularly, polyketide biosynthesis begins with a primerunit loading on to the active site of the condensing enzyme, β-keto acylsynthase (KS). An extender unit (usually malonate) is then transferredto the pathetheinyl arm of the acyl carrier protein (ACP). The KScatalyzes the condensation between the ACP-bound malonate and thestarter unit. Additional extender units are added sequentially until thenascent polyketide chain has grown to a desired chain length determinedby the protein chain length factor (CLF), perhaps together with the KS.Thus, the KS, CLF and the ACP form a minimal set to generate apolyketide backbone, and are together called the “minimal PKS.” Thenascent polyketide chain is then subjected to regiospecificketoreduction by a ketoreductase (KR) if it exists. Cyclases (CYC) andaromatases (ARO) later catalyze regiospecific ring formation eventsthrough intramolecular aldol condensations. The cyclized intermediatemay then undergo additional regiospecific and/or stereospecificmodifications (e.g., O-methylation, hydroxylation, glycosylation, etc.)controlled by downstream tailoring enzymes).

[0163] Acetyl CoA is the usual starter unit for most aromaticpolyketides. However, maloamyl CoA (Gatenbeck, S. Biochem. Biophy. Res.Commun. (1961) 6:422-426) and propionyl CoA (Paulick, R. C. et al. J.Am. Chem. Soc. (1976) 98:3370-3371) are primers for many members of thetetracycline and anthracycline classes of polyketides, respectively(FIG. 11). Daunorubicin PKS can also accept acetate, butyrate, andisobutyrate as starter units. (Oki, T. et al. J. Antibiot. (1981)34:783-790; Yoshimoto, A. et al. J. Antiobiot. (1993) 46:1758-1761).

[0164] The act KR can productively interact with all minimal PKSsstudied thus far and is both necessary and sufficient to catalyze a C-9ketoreduction. Although homologous KRs have been found in other PKSclusters, they catalyze ketoreduction with the same regiospecificity.However, the structures of frenolicin, griseusin and daunorubicin (FIG.11) suggest that an additional C-17 ketoreduction occurs in thesebiosynthetic pathways. Likewise, several angucyclines undergo a C-15ketoreduction, which occurs before the nascent polyketide chain iscyclized (Gould, S. J. et al. J. Am. Chem. Soc. (1992) 114:10066-10068).The ketoreductases responsible for C-15 and C-17 reductions have not yetbeen identified; however, two homologous KRs have been found in thedaunorubicin PKS cluster (Grimm, A. et al. Gene (1994) 151:1-10; Ye, J.et al. J. Bacteriol. (1994) 176:6270-6280). It is likely that theycatalyze the C-9 and C-17 reductions. Thus, KRs responsible forregiospecific reduction of the carbon chain backbone at positions otherthan C-9 may also be targets for use in the construction ofcombinatorial libraries.

[0165] The formation of the first two six-membered rings in thebiosynthesis of most naturally occurring bacterial aromatic polyketidesis controlled by PKS subunits; further ring closures are controlled byadditional cyclases and modifying enzymes. The structural diversityintroduced via these reactions appears to be greater than via the firsttwo cyclizations. However, certain preferred patterns are observed,which suggests that at least some of these downstream cyclases may beuseful for the construction of combinatorial libraries. For example, thepyran ring in isochromanequinones (FIG. 11) is invariably formed viacyclization between C-3 and C-15; two stereochemically distinct classesof products are observed (see, for example, the structures ofactinorhodin and frenolicin in (FIG. 11)). In anthracyclines andtetracyclines a third aldol condensation usually occurs between C-3 andC-16, whereas in unreduced tetracenomycins (FIG. 11) and relatedcompounds it occurs between C-5 and C-18, and in angucyclines (FIG. 11)it occurs between C-4 and C-17. Representative gene(s) encoding a few ofthese enzymes have already been cloned (Fernández-Moreno, M. A., et al.J. Biol. Chem. (1994) 269:24854-24863; Shen, B. et al. Biochemistry(1993) 32:11149-11154). At least some cyclases might recognize chains ofaltered lengths and/or degrees of reduction, thereby increasing thediversity of aromatic polyketide combinatorial libraries.

[0166] In the absence of downstream cyclases, polyketide chains undergonon-enzymatic reactions. Recently, some degree of predictability hasemerged within this repertoire of possibilities. For instance,hemiketals and benzene rings are two common moieties seen on the methylend. Hemiketals are formed with an appropriately-positioned enol and canbe followed by a dehydration. Benzene rings are formed with longeruncyclized methyl terminus. On the carboxyl terminus, a γ-pyrone ringformed by three ketide units is frequently observed. Spontaneousdecarboxylations occur on free carboxyl ends activated by the existenceof a β-carbonyl.

[0167] A cyclized intermediate can undergo various types ofmodifications to generate the final natural product. The recurrence ofcertain structural motifs among naturally occurring aromatic polyketidessuggests that some tailoring enzymes, particularly group transferases,may be combinatorially useful. Two examples are discussed below.

[0168] O-methylation is a common downstream modification. Althoughseveral SAM-dependent O-methyltransferase genes have been found in PKSgene clusters (Decker, H. et al. J. Bacteriol. (1993) 175:3876-3886),their specificities have not been systematically studied as yet. Perhapssome of them could be useful for combinatorial biosynthesis. Forinstance, O-11-methylation occurs in several members of theanthracycline, tetracenomycin, and angucycline classes of aromaticpolyketides (FIG. 11).

[0169] Both aromatic and complex polyketides are often glycosylated. Inmany cases (e.g. doxorubicin and erythromycin) absence of the sugargroup(s) results in considerably weaker bioactivity. There is tremendousdiversity in both the types and numbers of sugar units attached tonaturally occurring polyketide aglycones. In particular, deoxy- andaminosugars are commonly found. Regiochemical preferences can bedetected in many glycosylated natural products. Among anthracyclines,O-17 is frequently glycosylated, whereas among angucyclines, C-10 isusually glycosylated. Glycosyltransferases involved in erythromycinbiosynthesis may have relaxed specificities for the aglycone moiety(Donadio, S. et al. Science (1991) 252:675-679). An elloramycinglycosyltransferase may be able to recognize an unnatural NDP-sugar unitand attach it regiospecifically to an aromatic polyketide aglycone(Decker, H. et al. Angew. Chem. (1995), in press). These early resultssuggest that glycosyltransferases derived from secondary metabolicpathways have unique properties and may be attractive targets for use inthe generation of combinatorial libraries.

[0170] Although modular PKSs have not been extensively analyzed, theone-to-one correspondence between active sites and product structure(FIG. 9), together with the incredible chemical diversity observed amongnaturally occurring “complex” polyketides, indicates that thecombinatorial potential within these multienzyme systems could beconsiderably greater than that for aromatic PKSS. For example, a widerrange of primer units including aliphatic monomers (acetate, propionate,butyrate, isovalerate, etc.), aromatics (aminohydroxybenzoic acid),alicyclics (cyclohexanoic acid), and heterocyclics (pipecolic acid) arefound in various macrocyclic polyketides. Recent studies have shown thatmodular PKSs have relaxed specificity for their starter units (Kao etal. Science (1994), supra). The degree of β-ketoreduction following acondensation reaction can also be altered by genetic manipulation(Donadio et al. Science (1991), supra; Donadio, S. et al. Proc. Natl.Acad. Sci. USA (1993) 90:7119-7123). Likewise, the size of thepolyketide product can be varied by designing mutants with theappropriate number of modules (Kao, C. M. et al. J. Am. Chem. Soc.(1994) 116:11612-11613). Modular PKSs also exhibit considerable varietywith regards to the choice of extender units in each condensation cycle,although it remains to be seen to what extent this property can bemanipulated. Lastly, these enzymes are particularly well-known forgenerating an impressive range of asymmetric centers in their productsin a highly controlled manner. Thus, the combinatorial potential withinmodular PKS pathways could be virtually unlimited.

[0171] Like the actinomycetes, filamentous fungi are a rich source ofpolyketide natural products. The fact that fungal PKSs, such as the6-methylsalicylic acid synthase (6-MSAS) and the mevinolin synthase, areencoded by single multi-domain proteins (Beck et al. Eur. J. Biochem.(1990), supra; Davis, R. et al. Abstr. Genet. Ind. Microorg. Meeting,supra) indicates that they may also be targeted for combinatorialmutagenesis. Moreover, fungal PKSs can be functionally expressed in S.coelicolor CH999 using the genetic strategy outlined above. Chainlengths not observed in bacterial aromatic polyketides (e.g.tetraketides, pentaketides and hexaketides) have been found among fungalaromatic polyketides (O'Hagan, D. The Polyketide Metabolites (EllisHorwood, Chichester, U.K., 1991). Likewise, the cyclization patterns offungal aromatic polyketides are quite different from those observed inbacterial aromatic polyketides (Id.). In contrast with modular PKSs frombacteria, branched methyl groups are introduced into fungal polyketidebackbones by S-adenosylmethionine-dependent methyltransferases; in thecase of the mevinolin PKS (Davis, R. et al. Abstr. Genet. Ind. Microorg.Meeting, supra), this activity is encoded as one domain within amonocistronic PKS. It is now possible to experimentally evaluate whetherthese and other sources of chemical diversity in fungal polyketides areindeed amenable to combinatorial manipulation.

[0172] Based on the above-discussed state of the art, and the resultspresented in Examples 1-8 hereinbelow, the inventors herein havedeveloped the following set of design rules for rationally orstochastically manipulating early biosynthetic steps in aromaticpolyketide pathways including chain synthesis, C-9 ketoreduction, andthe formation of the first two aromatic rings. If each biosyntheticdegree of freedom was independent of all others, then it should bepossible to design a single combinatorial library of N₁×N₂× . . . N_(i)×. . . N_(n−1)×N_(n) clones, where N_(i) is the number of ways in whichthe ith degree of freedom can be exploited. In practice however, not allenzymatic degrees of freedom are independent. Therefore, to minimizeredundancy, it is preferable to design several sub-libraries of aromaticpolyketide-producing clones.

[0173] (1) Chain Length.

[0174] Polyketide carbon chain length is dictated by the minimal PKS(FIG. 12). Within the minimal PKS, the acyl carrier protein can beinterchanged without affecting specificity, whereas the chain lengthfactor is crucial. Although some ketosynthase/chain length factorcombinations are functional, others are not; therefore, biosynthesis ofa polyketide chain of specified length can be insured with a minimal PKSin which both the ketosynthase and chain length factor originate fromthe same PKS gene cluster. So far, chain lengths of 16 (octaketide), 18(nonaketide), 20 (decaketide), and 24 carbons (dodecaketide) can begenerated with minimal PKSs from the act, fren, tcm, and, whiE PKSclusters, respectively (McDaniel et al. Science (1993), supra; McDanielet al. J. Am. Chem. Soc. (1993), supra; McDaniel et al. Proc. Natl.Acad. Sci. USA (1994), supra). The whiE minimal PKS can also generate22-carbon backbones in the presence of a KR, suggesting a degree ofrelaxed chain length control as found for the fren PKS.

[0175] (2) Ketoreduction.

[0176] Ketoreduction requires a ketoreductase (FIG. 12). The act KR cancatalyze reduction of the C-9 carbonyl (counting from the carboxyl end)of a nascent polyketide backbone of any length studied so far.Furthermore, the act KR is compatible with all the minimal PKSsmentioned above. Homologous ketoreductases have been identified in otherPKS clusters (Sherman, D. H., et al. EMBO J. (1989) 8:2717-2725; Yu, T.-W. et al. J. Bacteriol. (1994) 176:2627-2534; Bibb, M. J. et al. Gene(1994) 142:31-39). These enzymes may catalyze ketoreduction at C-9 aswell since all the corresponding natural products undergo thismodification. In unusual circumstances, C-7 ketoreductions have alsobeen observed with the act KR.

[0177] (3) Cyclization of the First Ring.

[0178] Although the minimal PKS alone can control formation of the firstring, the regiospecific course of this reaction may be influenced byother PKS proteins. For example, most minimal PKSs studied so farproduce polyketides with C-7/C-12 cyclizations when present alone (FIG.12). In contrast, the tcm minimal PKS alone generates both C-7/C-12 andC-9/C-14 cyclized products. The presence of a ketoreductase with anyminimal PKS restricts the nascent polyketide chain to cyclizeexclusively with respect to the position of ketoreduction: C-7/C-12cyclization for C-9 ketoreduction and C-5/C-10 cyclization for C-7ketoreduction (McDaniel, R. et al. J. Am. Chem. Soc. (1993)115:11671-11675; McDaniel, R. et al. Proc. Natl. Acad. Sci. USA (1994)91:11542-11546; McDaniel, R. et al. J. Am. Chem. Soc. (1994)116:10855-10859). Likewise, use of the TcmN enzyme alters theregiospecificity to C-9/C-14 cyclizations for unreduced polyketides ofdifferent lengths, but has no effect on reduced molecules (see Example 5below).

[0179] (4) First Ring Aromatization.

[0180] The first ring in unreduced polyketides aromatizesnon-catalytically. In contrast, an aromatizing subunit is required forreduced polyketides (FIG. 12). There appears to be a hierarchy in thechain length specificity of these subunits from different PKS clusters.For example, the act ARO will recognize only 16-carbon chains (McDanielet al. Proc. Natl. Acad. Sci. USA (1994), supra), the fren AROrecognizes both 16- and 18-carbon chains, while the gris ARO recognizeschains of 16, 18, and 20 carbons.

[0181] (5) Second Ring Cyclization.

[0182] C-5/C-14 cyclization of the second ring of reduced polyketidesmay be achieved with an appropriate cyclase (FIG. 12). While the act CYCcan cyclize octa- and nonaketides, it does not recognize longer chains.No equivalent C-5/C-14 cyclase with specificity for decaketides orlonger chains has been identified, although the structures of naturalproducts such as griseusin imply their existence. In the case ofsufficiently long unreduced chains with a C-9/C-14 first ring, formationof a C-7/C-16 second ring is catalyzed by the minimal PKS (FIG. 12)(McDaniel et al. Proc. Natl. Acad. Sci. USA (1994), supra).

[0183] (6) Additional Cyclizations.

[0184] The KS, CLF, ACP, KR, ARO, and CYC subunits of the PKS togethercatalyze the formation of an intermediate with a defined chain length,reduction pattern, and first two cyclizations. While the biosynthesis ofnaturally occurring polyketides typically requires the activity ofdownstream cyclases and other modifying enzymes to generate thecharacteristic biologically active product, subsequent reactions in thebiosynthesis of engineered polyketides described here and in our earlierwork occur in the absence of specific enzymes and are determined by thedifferent physical and chemical properties of the individual molecules.Presumably reflecting such chemical possibilities and constraints,consistent patterns have been observed, leading to some degree ofpredictability. Two common moieties formed by the uncyclized methylterminus of polyketide chains are hemiketals and benzene rings.Formation of a hemiketal occurs in the presence of an appropriatelypositioned enol and can be followed by a dehydration since both thehydrated and dehydrated forms are often isolated (FIG. 13(a)) (McDaniel,R. et al. Science (1993) 262:15461550; McDaniel, R. et al. J. Am. Chem.Soc. (1994) 116:10855-10859; Fu, H. et al. J. Am. Chem. Soc. (1994)116:4166-4170), while benzene ring formation occurs with longerunprocessed methyl ends (FIG. 13(b)) (Fu et al. J. Am. Chem. Soc.(1994), supra). The most frequently observed moiety at the carboxylterminus of the chain is a γ-pyrone ring formed by three ketide units(FIG. 13(c)) (McDaniel et al. J. Am. Chem. Soc. (1994), supra; Fu et al.J. Am. Chem. Soc. (1994), supra; Fu, H., et al. Biochemistry (1994)33:9321-9326; Fu, H. et al. Chem. & Biol. (1994) 1:205-210; Zhang, H.-l. et al. J. Org. Chem. (1990) 55:1682-1684); if a free carboxylic acidremains, decarboxylation typically occurs if a β-carbonyl exists (FIG.13(d)) (McDaniel et al. Science (1993), supra; McDaniel, R.,Ebert-Khosla, S., Hopwood, D. A. & Khosla, C. J. Am. Chem. Soc. (1993),supra; Kao, C. M. et al. J. Am. Chem. Soc. (1994) 116:11612-11613). Manyaldol condensations can be predicted as well, bearing in mind that themethyl and carboxyl ends tend preferentially to cyclize independentlybut will co-cyclize if no alternative exists (FIG. 13(e)) (McDaniel etal. Proc. Natl. Acad. Sci. USA (1994), supra. These non-enzymaticcyclization patterns observed in vivo are also consistent with earlierbiomimetic studies (Griffin, D. A. et al. J. Chem. Soc. Perkin Trans.(1984) 1:1035-1042).

[0185] Taken together with the structures of other naturally occurringbacterial aromatic polyketides, the design rules presented above can beextrapolated to estimate the extent of molecular diversity that might begenerated via in vivo combinatorial biosynthesis of, for example,reduced and unreduced polyketides. For reduced polyketides, theidentified degrees of freedom include chain length, aromatization of thefirst ring, and cyclization of the second ring. For unreduced ones,these include chain length and regiospecificity of the first ringcyclization. The number of accessible structures is the product of thenumber of ways in which each degree of freedom can be varied. Chains offive different lengths have so far been manipulated (16-, 18-20-, 22-and 24-carbon lengths). From the structure and deduced biosyntheticpathways of the dynemicin anthraquinone (Tokiwa, Y. et al. J. Am. Chem.Soc. (1992) 114:4107-4110), simaomicin (Carter, G. T. et al. J. Org.Chem. (1989) 54:4321-4323), and benastatin (Aoyama, T. et al. J.Antibiot. (1992) 45:1767-1772), the isolation of minimal PKSs thatgenerate 14-, 26-, and possibly 28-carbon backbones, respectively, isanticipated, bringing the potential number to eight. Cloning of suchminimal PKSs can be accomplished using the genes for minimal PKSs whichhave previously been isolated, such as the actI genes (Sherman et al.EMBO J. (1989), supra; Yu et al. J. Bacteriol. (1994), supra; Bibb etal. Gene (1994), supra; Malpartida, F. et al. Nature (1987)325:818-821). Reduced chains can either be aromatized or not; a secondring cyclase is optional where the first ring is aromatized (FIG. 12).The regiospecificity of the first cyclization of an unreduced chain canbe varied, depending on the presence of an enzyme like TcmN.

[0186] For example, for reduced polyketides the relevant degrees offreedom include the chain length (which can be manipulated in at leastseven ways), the first ring aromatization (which can be manipulated inat least two ways), and the second ring cyclization (which can bemanipulated in at least two ways for aromatized intermediates only). Forunreduced polyketides, the regiospecificity of the first cyclization canalso be manipulated. Thus, the combinatorial potential for reducedpolyketides is at least 7×3=21; for unreduced polyketides thecombinatorial potential is at least 7×2=14. Moreover, these numbers donot include additional minor products, on the order of 5 to 10 per majorproduct, that are produced in the recombinant strains throughnon-enzymatic or non-specific enzyme catalyzed steps. Thus, the numberof polyketides that can be generated from combinatorial manipulation ofonly the first few steps in aromatic polyketide biosynthesis is on theorder of a few hundred. Thus, genetically engineered biosynthesisrepresents a potentially unlimited source of chemical diversity for drugdiscovery.

[0187] The number of potential novel polyketides increase geometricallyas new degrees of freedom are exploited and/or protein engineeringstrategies are brought to bear on the task of creating enzyme subunitswith specificities not observed in nature. For example, non-acetatestarter units can be incorporated into polyketide backbones (e.g.propionate in daunorubicin and malonamide in oxytetracycline).Furthermore, enzymes that catalyze downstream cyclizations and late-stepmodifications, such as group transfer reactions and oxidoreductionscommonly seen in naturally occurring polyketides, can be studied alongthe lines presented here and elsewhere. It is therefore possible that atleast some of these degrees of freedom can be combinatorially exploitedto generate libraries of synthetic products with structural diversitythat is comparable to that observed in nature.

[0188] C. Experimental

[0189] Below are examples of specific embodiments for carrying out thepresent invention. The examples are offered for illustrative purposesonly, and are not intended to limit the scope of the present inventionin any way.

[0190] Efforts have been made to ensure accuracy with respect to numbersused (e.g., amounts, temperatures, etc.), but some experimental errorand deviation should, of course, be allowed for.

Materials and Methods

[0191] Bacterial Strains, Plasmids, and Culture Conditions.

[0192]S. coelicolor CH999 was used as a host for transformation by allplasmids. The construction of this strain is described below. DNAmanipulations were performed in Escherichia coli MC1061. Plasmids werepassaged through E. coli ET12567 (dam dcm hsds Cm^(r)) (MacNeil, D. J.J. Bacteriol. (1988) 170:5607) to generate unmethylated DNA prior totransformation of S. coelicolor. E. coli strains were grown understandard conditions. S. coelicolor strains were grown on R2YE agarplates (Hopwood, D. A. et al. Genetic manipulation of Streptomyces. Alaboratory manual. The John Innes Foundation: Norwich, 1985).

[0193] Manipulation of DNA and Organisms.

[0194] Polymerase chain reaction (PCR) was performed using Taqpolymerase (Perkin Elmer Cetus) under conditions recommended by theenzyme manufacturer. Standard in vitro techniques were used for DNAmanipulations (Sambrook, et al. Molecular Cloning: A Laboratory Manual(Current Edition)). E. coli was transformed with a Bio-Rad E. ColiPulsing apparatus using protocols provided by Bio-Rad. S. coelicolor wastransformed by standard procedures (Hopwood, D. A. et al. Geneticmanipulation of Streptomyces. A laboratory manual. The John InnesFoundation: Norwich, 1985) and transformants were selected using 2 ml ofa 500 mg/ml thiostrepton overlay.

[0195] Construction of Plasmids Containing Recombinant PKSs.

[0196] All plasmids are derivatives of pRM5, described below. fren PKSgenes were amplified via PCR with 5′ and 3′ restriction sites flankingthe genes in accordance with the location of cloning sites on pRM5 (i.e.PacI-NsiI for ORF1, NsiI-XbaI for ORF2, and XbaI-PstI for ORF3).Following subcloning and sequencing, the amplified fragments were clonedin place of the corresponding fragments in pRM5 to generate the plasmidsfor transformation.

[0197] Production and Purification of Polyketides.

[0198] For initial screening, all strains were grown at 30° C. asconfluent lawns on 10-30 plates each containing approximately 30 ml ofagar medium for 6-8 days. Additional plates were made as needed toobtain sufficient material for complete characterization. CH999 was anegative control when screening for potential polyketides. The agar wasfinely chopped and extracted with ethyl acetate/1% acetic acid or ethylacetate:methanol (4:1)/1% acetic acid. The concentrated extract was thenflashed through a silica gel (Baker 40 mm) chromatography column inethyl acetate/1% acetic acid. Alternatively, the extract was applied toa Florisil column (Fisher Scientific) and eluted with ethylacetate:ethanol:acetic acid (17:2:1). The primary yellow fraction wasfurther purified via high-performance liquid chromatography (HPLC) usinga 20-60% acetonitrile/water/1% acetic acid gradient on a preparativereverse phase (C-18) column (Beckman). Absorbance was monitored at 280nm and 410 nm. In general, the yield of purified product from thesestrains was approximately 10 mg/l for compounds 1 and 2 (FIG. 4), and 5mg/l for compounds 7 and 8 (FIG. 7).

[0199] SEK4, (12), was produced and purified as follows. CH999/pSEK4 wasgrown on 90 agar plates (˜34 ml/plate) at 30° C. for 7 days. The agarwas chopped and extracted with ethyl acetate/methanol (4/1) in thepresence of 1% acetic acid (3×1000 ml). Following removal of the solventunder vacuum, 200 ml of ethyl acetate containing 1% acetic acid wereadded. The precipitate was filtered and discarded, and the solvent wasevaporated to dryness. The product mixture was applied to a Florisilcolumn (Fisher Scientific), and eluted with ethyl acetate containing 3%acetic acid. The first 100 ml fraction was collected, and concentrateddown to 5 ml. 1 ml methanol was added, and the mixture was kept at 4° C.overnight. The precipitate was collected by filtration, and washed withethyl acetate to give 850 mg of pure product. R_(f)=0.48 (ethyl acetatewith 1% acetic acid). Results from NMR spectroscopy on SEK4 are reportedin Table 4. FAB HRMS (NBA), M+H⁺, calculated m/e 319.0818, observed m/e319.0820.

[0200] To produce SEK15 (13) and SEK15b (16), CH999/pSEK15 was grown on90 agar plates, and the product was extracted in the same manner asSEK4. The mixture was applied to a Florisil column (ethyl acetate with5% acetic acid), and fractions containing the major products werecombined and evaporated to dryness. The products were further purifiedusing preparative C-18 reverse phase HPLC (Beckman) (mobile phase:acetonitrile/water=1/10 to 3/5 gradient in the presence of 1% aceticacid). The yield of SEK15, (13), was 250 mg. R_(f)=0.41 (ethyl acetatewith 1% acetic acid). Results from NMR spectroscopy on SEK4 are reportedin Table 4. FAB HRMS (NBA), M+H⁺, calculated m/e 385.0923, observed m/e385.0920.

[0201] [1,2-¹³C₂] Acetate Feeding Experiments.

[0202] Two 2 l flasks each containing 400 ml of modified NMP medium(Strauch, E. et al. Mol. Microbiol. (1991) 5:289) were inoculated withspores of S. coelicolor CH999/pRM18, CH999/pSEK4 or CH999/pSEK15, andincubated in a shaker at 30 degrees C. and 300 rpm. To each flask, 50 mgof sodium [1,2-¹³C₂] acetate (Aldrich) was added at 72 and 96 hrs. After120 hrs, the cultures were pooled and extracted with two 500 ml volumesof ethyl acetate/1% acetic acid. The organic phase was kept andpurification proceeded as described above. ¹³C NMR data indicateapproximately a 2-3% enrichment for the CH999/pRM18 product; a 0.5-1%enrichment for SEK4 and a 1-2% enrichment for SEK15.

[0203] NMR Spectroscopy.

[0204] All spectra were recorded on a Varian XL-400 except for HETCORanalysis of RM18 (10) (FIG. 8), which was performed on a Nicolet NT-360.¹³C spectra were acquired with continuous broadband proton decoupling.For NOE studies of RM18 (10), the one-dimensional difference method wasemployed. All compounds were dissolved in DMSO-d₆ (Sigma, 99+atom % D)and spectra were referenced internally to the solvent. Hydroxylresonances were identified by adding D₂O (Aldrich, 99 atom % D) andchecking for disappearance of signal.

EXAMPLE 1 Production of S. coelicolor CH999

[0205] An S. coelicolor host cell, genetically engineered to remove thenative act gene cluster, and termed CH999, was constructed using S.coelicolor CH1 (Khosla, C. Molec. Microbiol. (1992) 6:3237), using thestrategy depicted in FIG. 2. (CH1 is derived from S. coelicolor B385(Rudd, B. A. M. Genetics of Pigmented Secondary Metabolites inStreptomyces coelicolor (1978) Ph.D. Thesis, University of East Anglia,Norwich, England.) CH1 includes the act gene cluster which codes forenzymes involved in the biosynthesis and export of the polyketideantibiotic actinorhodin. The cluster is made up of the PKS genes,flanked by several post-PKS biosynthetic genes including those involvedin cyclization, aromatization, and subsequent chemical tailoring (FIG.2A). Also present are the genes responsible for transcriptionalactivation of the act genes. The act gene cluster was deleted from CH1using homologous recombination as described in Khosla, C. et al. Molec.Microbiol. (1992) 6:3237.

[0206] In particular, plasmid pLRermEts (FIG. 2B) was constructed withthe following features: a ColEI replicon from pBR322, the temperaturesensitive replicon from PSG5 (Muth, G. et al. Mol. Gen. Genet. (1989)219:341), ampicillin and thiostrepton resistance markers, and adisruption cassette including a 2 kb BamHI/XhoI fragment from the 5′ endof the act cluster, a 1.5 kb ermE fragment (Khosla, C. et al. Molec.Microbiol. (1992) 6:3237), and a 1.9 kb SphI/PstI fragment from the 3′end of the act cluster. The 5′ fragment extended from the BamHI site 1(Malpartida, F. and Hopwood, D. A. Nature (1984) 309:462; Malpartida, F.and Hopwood, D. A. Mol. Gen. Genet. (1986) 205:66) downstream to a XhoIsite. The 3′ fragment extended from PstI site 20 upstream to SphI site19.2 (Fernandez-Moreno, M. A. et al. J. Biol. Chem. (1992) 267:19278).The 5′ and 3′ fragments (shown as hatched DNA in FIG. 2) were cloned inthe same relative orientation as in the act cluster. CH1 was transformedwith pLRermEts. The plasmid was subsequently cured from candidatetransformants by streaking non-selectively at 39° C. Several coloniesthat were lincomycin resistant, thiostrepton sensitive, and unable toproduce actinorhodin, were isolated and checked via Southern blotting.One of them was designated CH999.

EXAMPLE 2 Production of the Recombinant Vector pRM5

[0207] Shuttle plasmids are used to express recombinant PKSs in CH999.Such plasmids typically include a colEI replicon, an appropriatelytruncated SCP2* Streptomyces replicon, two act-promoters to allow forbidirectional cloning, the gene encoding the actII-ORF4 activator whichinduces transcription from act promoters during the transition fromgrowth phase to stationary phase, and appropriate marker genes.Restriction sites have been engineered into these vectors to facilitatethe combinatorial construction of PKS gene clusters starting fromcassettes encoding individual subunits (or domains) of naturallyoccurring PKSS. The primary advantages of this method are that (i) allrelevant biosynthetic genes are plasmid-borne and therefore amenable tofacile manipulation and mutagenesis in E. coli, (ii) the entire libraryof PKS gene clusters can be expressed in the same bacterial host whichis genetically and physiologically well-characterized and presumablycontains most, if not all, ancillary activities required for in vivoproduction of polyketides, (iii) polyketides are produced in a secondarymetabolite-like manner, thereby alleviating the toxic effects ofsynthesizing potentially bioactive compounds in vivo, and (iv) moleculesthus produced undergo fewer side reactions than if the same pathwayswere expressed in wild-type organisms or blocked mutants. pRM5 (FIG. 3)was the shuttle plasmid used for expressing PKSs in CH999. It includes aColEI replicon to allow genetic engineering in E. coli, an appropriatelytruncated SCP2* (low copy number) Streptomyces replicon, and theactII-ORF4 activator gene from the act cluster, which inducestranscription from act promoters during the transition from growth phaseto stationary phase in the vegetative mycelium. As shown in FIG. 3, pRM5carries the divergent actI/actIII promoter pair, together withconvenient cloning sites to facilitate the insertion of a variety ofengineered PKS genes downstream of both promoters. pRM5 lacks the parlocus of SCP2*; as a result the plasmid is slightly unstable (approx. 2%loss in the absence of thiostrepton). This feature was deliberatelyintroduced in order to allow for rapid confirmation that a phenotype ofinterest could be unambiguously assigned to the plasmid-borne mutantPKS. The recombinant PKSs from pRM5 are expressed approximately at thetransition from exponential to stationary phase of growth, in goodyields.

[0208] pRM5 was constructed as follows. A 10.5 kb SphI/HindIII fragmentfrom pIJ903 (containing a portion of the fertility locus and the originof replication of SCP2* as well as the colEI origin of replication andthe β-lactamase gene from pBR327) (Lydiate, D. J. Gene (1985) 35:223)was ligated with a 1.5 kb HindIII/SphI tsr gene cassette to yield pRM1.pRM5 was constructed by inserting the following two fragments betweenthe unique HindIII and EcoRI sites of pRM1: a 0.3 kb HindIII/HpaI(blunt)fragment carrying a transcription terminator from phage fd (Khosla, C.et al. Molec. Microbiol. (1992) 6:3237), and a 10 kb fragment from theact cluster extending from the NcoI site (1 kb upstream of theactII-ORF4 activator gene) (Hallam, S. E. et al. Gene (1988) 74:305;Fernandez-Moreno, M. A. et al. Cell (1991) 66:769; Caballero, J. L. Mol.Gen. Genet. (1991) 230:401) to the PstI site downstream of theactI-VII-IV genes (Fernandez-Moreno, M. A. et al. J. Biol. Chem. (1992)267:19278).

[0209] To facilitate the expression of any desired recombinant PKS underthe control of the actI promoter (which is activated by the actII-ORF4gene product), restriction sites for PacI, NsiI, XbaI, and PstI wereengineered into the act DNA in intercistronic positions. In pRM5, aswell as in all other PKS expression plasmids described here, ORF1, 2,and 3 alleles were cloned between these sites as cassettes engineeredwith their own RBSs.

[0210] In particular, in most naturally occurring aromatic polyketidesynthase gene clusters in actinomycetes, ORF1 and ORF2 aretranslationally coupled. In order to facilitate construction ofrecombinant PKSs, the ORF1 and ORF2 alleles used here were cloned asindependent (uncoupled) cassettes. For act ORF1, the following sequencewas engineered into pRM5: CCACCGGACGAACGCATCGATTAATTAAGGAGGACCATCATG,where the boldfaced sequence corresponds to upstream DNA from the actIregion, TTAATTAA is the PacI recognition site, and ATG is the startcodon of act ORF1. The following sequence was engineered between actORF1 and ORF2: NTGAATGCATGGAGGAGCCATCATG, where TGA and ATG are the stopand start codons of ORF1 and ORF2, respectively, ATGCAT is the NsiIrecognition site, and the replacement of N (A in act DNA, A or G inalleles from other PKSs) with a C results in translational decoupling.The following sequence was engineered downstream of act ORF2: TAATCTAGA,where TAA is the stop codon, and TCTAGA is the XbaI recognition site.This allowed fusion of act ORF1 and ORF2 (engineered as above) to anXbaI site that had been engineered upstream of act ORF3 (Khosla, C. etal. Molec. Microbiol. (1992) 6:3237). As a control, pRM2 wasconstructed, identical to pRM5, but lacking any of the engineeredsequences. ORF1 and ORF2 in pRM2 are translationally coupled. Comparisonof the product profiles of CH999/pRM2 and CH999/pRM5 revealed that thedecoupling strategy described here had no detectable influence onproduct distribution or product levels.

EXAMPLE 3 Polyketides Produced Using CH999 Transformed with pRM5

[0211] Plasmid pRM5 was introduced into S. coelicolor CH999 usingstandard techniques. (See, e.g., Sambrook, et al. Molecular Cloning: ALaboratory Manual (Current Edition.) CH999 transformed with pRM5produced a large amount of yellowish-brown material. The two mostabundant products were characterized by NMR and mass spectroscopy asaloesaponarin II (2) (Bartel, P. L. et al. J. Bacteriol. (1990)172:4816) and its carboxylated analog,3,8-dihydroxy-1-methylanthraquinone-2-carboxylic acid (1) (Cameron, D.W. et al. Liebigs Ann. Chem. (1989) 7:699) (FIG. 4). It is presumed that2 is derived from 1 by non-enzymatic decarboxylation (Bartel, P. L. etal. J. Bacteriol. (1990) 172:4816). Compounds 1 and 2 were present inapproximately a 1:5 molar ratio. Approximately 100 mg of the mixturecould be easily purified from 1 l of culture. The CH999/pRM5 host-vectorsystem was therefore functioning as expected to produce significantamounts of a stable, only minimally modified polyketide metabolite. Theproduction of 1 and 2 is consistent with the proposed pathway ofactinorhodin biosynthesis (Bartel, P. L. et al. J. Bacteriol. (1990)172:4816). Both metabolites, like the actinorhodin backbone, are derivedfrom a 16-carbon polyketide with a single ketoreduction at C-9.

[0212] When CH999 was transformed with pSEK4, identical to pRM5 exceptfor replacement of a 140 bp SphI/SalI fragment within the act KR gene bythe SphI/SalI fragment from pUC19, the resulting strain producedabundant quantities of the aromatic polyketide SEK4 (12). The exactstructure of this product is slightly different from desoxyerythrolaccin(Bartel, P. L. et al. J. Bacteriol. (1990) 172:4816). However, in vivoisotopic labeling studies using 1,2-¹³C₂- labeled acetate confirmed thatthe polyketide backbone is derived from 8 acetates. Moreover, thearomatic region of the ¹H spectrum, as well as the ¹³C NMR spectrum ofthis product, are consistent with a tricyclic structure similar to 1,but lacking any ketoreduction (see Table 4).

EXAMPLE 4 Construction and Analysis of Hybrid Polyketide Synthases

[0213] A. Construction of Hybrid PKSs Including Components from act, graand tcm PKSs

[0214]FIG. 1A shows the PKSs responsible for synthesizing the carbonchain backbones of actinorhodin (3), granaticin (4), and tetracenomycin(5) (structures shown in FIG. 5) which contain homologous putative KS/ATand ACP subunits, as well as the ORF2 product. The act and gra PKSs alsohave KRs, lacking in the tcm PKS. Corresponding proteins from eachcluster show a high degree of sequence identity. The percentageidentities between corresponding PKS proteins in the three clusters areas follows: KS/AT: act/gra 76, act/tcm 64, gra/tcm 70; CLF: act/gra 60,act/tcm 58, gra/tcm 54; ACP: act/gra 60, act/tcm 43, gra/tcm 44. The actand gra PKSS synthesize identical 16-carbon backbones derived from 8acetate residues with a ketoreduction at C-9 (FIG. 6). In contrast, alsoas shown in FIG. 6, the tcm polyketide backbone differs in overallcarbon chain length (20 instead of 16 carbons), lack of anyketoreduction, and regiospecificity of the first cyclization, whichoccurs between carbons 9 and 14, instead of carbons 7 and 12 for act andgra.

[0215] In an attempt to generate novel polyketides, differing in a rangeof properties, as well as to elucidate aspects of the programming ofaromatic PKSs, a systematic series of minimal PKS gene clusters, usingvarious permutations of the ORF1 (encoding the KS/AT subunit), ORF2(encoding the CLF subunit) and ORF3 (encoding the ACP subunit) geneproducts from the act, gra and tcm gene clusters were cloned into pRM5in place of the existing act genes, as shown in Table 1. The resultingplasmids were used to transform CH999 as above.

[0216] Analysis of the products of the recombinant PKSs containingvarious permutations among the KS/AT, ORF2 product, and ACP subunits ofthe PKSs (all constructs also containing the act KR, cyclase, anddehydratase genes) indicated that the synthases could be grouped intothree categories (Table 1): those that did not produce any polyketide;those that produced compound 1 (in addition to a small amount of 2); andthose that produced a novel polyketide 9 (designated RM20) (FIG. 6). Thestructure of 9 suggests that the polyketide backbone precursor of thismolecule is derived from 10 acetate residues with a single ketoreductionat the C-9 position.

[0217] In order to investigate the influence of the act KR on thereduction and cyclization patterns of a heterologous polyketide chain,pSEK15 was also constructed, which included tcm ORFs 1-3, but lacked theact KR. (The deletion in the act KR gene in this construct was identicalto that in pSEK4.) Analysis of CH999/pSEK15 showed the 20 carbon chainproduct, SEK15 (13) which resembled, but was not identical to,tetracenomycin C or its shunt products. NMR spectroscopy was alsoconsistent with a completely unreduced decaketide backbone (see Table4).

[0218] All act/gra hybrids produced compound 1, consistent with theidentical structures of the presumed actinorhodin and granaticinpolyketides. In each case where a product could be isolated from atcm/act hybrid, the chain length of the polyketide was identical to thatof the natural product corresponding to the source of ORF2. This impliesthat the ORF2 product, and not the ACP or KS/AT, controls carbon chainlength. Furthermore, since all polyketides produced by the hybridsdescribed here, except the ones lacking the KR (CH999/pSEK4 andCH999/pSEK15), underwent a single ketoreduction, it can be concludedthat: (i) the KR is both necessary and sufficient for ketoreduction tooccur; (ii) this reduction always occurs at the C-9 position in thefinal polyketide backbone (counting from the carboxyl end of the chain);and (iii) while unreduced polyketides may undergo alternativecyclization patterns, in nascent polyketide chains that have undergoneketoreduction, the regiochemistry of the first cyclization is dictatedby the position of the resulting hydroxyl, irrespective of how thiscyclization occurs in the non-reduced product. In other words, the tcmPKS could be engineered to exhibit new cyclization specificity byincluding a ketoreductase.

[0219] A striking feature of RM20 (9) is the pattern of cyclizationsfollowing the first cyclization. Isolation of mutactin (6) from anactVII mutant suggested that the actVII product and its tcm homologcatalyze the cyclization of the second ring in the biosynthesis ofactinorhodin (3) and tetracenomycin (5), respectively (Sherman, D. H. etal. Tetrahedron (1991) 47:6029; Summers, R. G. et al. J. Bacteriol.(1992) 174:1810). The cyclization pattern of RM20 (9) is different fromthat of 1 and tetracenomycin F1, despite the presence of the actVII geneon pRM20 (9). It therefore appears that the act cyclase cannot cyclizelonger polyketide chains.

[0220] Unexpectedly, the strain containing the minimal tcm PKS alone(CH999/pSEK33) produced two polyketides, SEK15 (13) and SEK15b (16), asdepicted in FIG. 8, in approximately equal quantities. Compounds (13)and (16) were also isolated from CH999/pSEK15, however, greaterquantities of compound (13) were isolated this construct than ofcompound (16).

[0221] SEK15b is a novel compound, the structure of which was elucidatedthrough a combination of NMR spectroscopy, sodium [1,2-¹³C₂] acetatefeeding experiments and mass spectroscopy. Results from ¹H and ¹³C NMRindicated that SEK15b consisted of an unreduced anthraquinone moiety anda pyrone moiety. Sodium [1,2-¹³C₂]-acetate feeding experiments confirmedthat the carbon chain of SEK15b was derived from 10 acetate units. Thecoupling constants calculated from the ¹³C NMR spectrum of the enrichedSEK15b sample facilitated peak assignment. Fast atom bombardment(FAB)-mass spectroscopy gave a molecular weight of 381 (M+H⁺),consistent with C₂₀H₁₂O₈. Deuterium exchange was used to confirm thepresence of each hydroxyl in SEK15b.

[0222] In order to identify the degrees of freedom available in vivo toa nascent polyketide chain for cyclizing in the absence of an activecyclase, polyketides produced by recombinant S. coelicolor CH999/pRM37(McDaniel et al. (1993), supra) were analyzed. The biosynthetic enzymesencoded by pRM37 are the tcm ketosynthase/acyltransferase (KS/AT), thetcm chain length determining factor (CLF), the tcm acyl carrier protein(ACP), and the act ketoreductase (KR).

[0223] Two novel compounds, RM20b (14) and RM20c (15) (FIG. 8) werediscovered in the culture medium of CH999/pRM37, which had previouslyyielded RM20 (9). The relative quantities of the three compoundsrecovered were 3:7:1 (RM20:RM20b:RM20c). The structures of (14) and (15)were elucidated through a combination of mass spectroscopy, NMRspectroscopy and isotope labeling experiments. ¹H and ¹³C NMR spectrasuggested that RM20b and RM20c were diastereomers, each containing apyrone moiety. Optical rotations ([α]_(D) ²⁰ were found to by +210.8°for RM20b (EtOH, 0.55%) and +78.0° for RM20c (EtOH, 0.33%). Sodium[1,2-¹³C₂]-acetate feeding experiments confirmed that the carbon chainof RM20b (and by inference RM20c) was derived from 10 acetate units.Deuterium exchange studies were carried out in order to identify ¹H NMRpeaks corresponding to potential hydroxyl groups on both RM20b andRM20c. Proton coupling constants were calculated from the results of ¹HNMR and one-dimensional decoupling experiments. In particular, thecoupling pattern in the upfield region of the spectrum indicated a5-proton spin system of two methylene groups surrounding a centralcarbinol methine proton. High resolution fast atom bombardment (FAB)mass spectroscopy gave molecular weights of (519.0056) (M=Cs⁺) for RM20band 387.1070 (M+H⁺) for RM20c, which is consistent with C₂₀H₁₈O₈ (M+Cs⁺,519.0056; M+H⁺, 387.1080). Based on theses data, structures (14) and(15) (FIG. 8) were assigned to RM20b and RM20c, respectively.

[0224] Data from ¹H and ¹³C NMR indicated that the coupling constantsbetween H-9 and the geminal protons on C-8 were 12.1 or 12.2 and 2.5 or2.2 Hz for RM20b or RM20c, respectively. The coupling constants betweenH-9 and the geminal protons on C-10 were 9.6 or 9.7 and 5.7 or 5.8 Hzfor Rm20b or RM20c, respectively. These values are typical of a J_(a,a)(j_(9a,8a) or J_(9a,10a)) and J_(a,e) (J_(9a,8e) or J_(9a,10e)) couplingpattern, and indicate an axial position for H-9 in both RM20b and Rm20c.In contrast, the chemical shifts of the C-7 hydroxyls on the twomolecules were 16.18 and 6.14 ppm for RM20b and RM20c, respectively.These values indicate a hydrogen bond between the C-7 hydroxyl and asuitably positioned acceptor atom in RM20b, but not in RM20c. The mostlikely candidate acceptor atoms for such hydrogen bonding are the C-13carbonyl oxygen in the conjugated pyrone ring system, or the bridgeoxygen in the isolate pyrone ring. The former appears to be likely as itwould be impossible to discriminate between (14) and (15) if the latterwere the case. Furthermore, comparison of ¹³C NMR spectra of RM20b andRM20c revealed that the greatest differences between (14) and (15) werein the chemical shifts of the carbons that make up the conjugated pyronering (+5.9, −6.1, +8.9, −7.8 and +2.0 ppm for C-11, C-12, C-13, C-14 andC-15, respectively). Such a pattern of alternating upfield and downfieldshifts can be explained by the fact that the C-7 hydroxyl ishydrogen-bonded to the C-13 carbonyl, since hydrogen bonding would beexpected to reduce the electron density around C-11, C-13 and C-15, butincrease the electron density around C-12 and C-14. To confirm theC-7/C-13 hydrogen bond assignment, the exchangeable protons RM20b andRM20c were replaced with deuterium (by incubating in the presence ofD₂O), and the samples were analyzed by ¹³C NMR. The C-13 peak in RM20b,but not RM20c, underwent an upfield shift (1.7 ppm), which can beexplained by a weaker C-7/C-13 non-covalent bond in RM20b when hydrogenis replace with deuterium. In order to form a hydrogen bond with theC-13 carbonyl, the C-7 hydroxyl of RM20b must occupy the equatorialposition. Thus, it can be inferred that the C-7 and C-9 hydroxyls are onthe same face (syn) of the conjugated ring system in the major isomer(RM20b), whereas they are on opposite sides (anti) in the minor isomer(RM20c).

[0225] No polyketide could be detected in CH999/pRM15, /pRM35, and/pRM36. Thus, only some ORF1-ORF2 combinations are functional. Sinceeach subunit was functional in at least one recombinant synthase,protein expression/folding problems are unlikely to be the cause.Instead, imperfect or inhibitory association between the differentsubunits of these enzyme complexes, or biosynthesis of (aborted) shortchain products that are rapidly degraded, are plausible explanations.

[0226] B. Construction of Hybrid PKSs Including Components from act andfren PKSs

[0227]Streptomyces roseofulvus produces both frenolicin B (7) (Iwai, Y.et al. J. Antibiot. (1978) 31:959) and nanaomycin A (8) (Tsuzuki, K. etal. J. Antibiot. (1986) 39:1343). A 10 kb DNA fragment (referred to asthe fren locus hereafter) was cloned from a genomic library of S.roseofulvus (Bibb, M. J. et al. submitted) using DNA encoding the KS/ATand KR components of the act PKS of S. coelicolor A3(2) as a probe(Malpartida, F. et al. Nature (1987) 325:818). (See FIG. 7 forstructural representations.) DNA sequencing of the fren locus revealedthe existence of (among others) genes with a high degree of identity tothose encoding the act KS/AT, CLF, ACP, KR, and cyclase.

[0228] To produce the novel polyketides, the ORF1, 2 and 3 act genespresent in pRM5 were replaced with the corresponding fren genes, asshown in Table 2. S. coelicolor CH999, constructed as described above,was transformed with these plasmids. (The genes encoding the act KR, andthe act cyclase were also present on each of these genetic constructs.)Based on results from similar experiments with act and tcm PKSs,described above, it was expected that the act KR would be able to reducethe products of all functional recombinant PKSs, whereas the ability ofthe act cyclase to catalyze the second cyclization would depend upon thechain length of the product of the fren PKS.

[0229] The results summarized in Table 2 indicate that most of thetransformants expressed functional PKSs, as assayed by their ability toproduce aromatic polyketides. Structural analysis of the major productsrevealed that the producer strains could be grouped into two categories:those that synthesized compound 1 (together with a smaller amount of itsdecarboxylated side-product (2), and those that synthesized a mixture ofcompounds 1, 10 and 11 in a roughly 1:2:2 ratio. (Small amounts of 2were also found in all strains producing 1.) Compounds 1 and 2 had beenobserved before as natural products, and were the metabolites producedby a PKS consisting entirely of act subunits, as described in Example 3.Compounds 10 and 11 (designated RM18 and RM18b, respectively) are novelstructures whose chemical synthesis or isolation as natural products hasnot been reported previously.

[0230] The structures of 10 and 11 were elucidated through a combinationof mass spectroscopy, NMR spectroscopy, and isotope labelingexperiments. The ¹H and ¹³C spectral assignments are shown in Table 3,along with ¹³C-¹³C coupling constants for 10 obtained through sodium[1,2-¹³C₂] acetate feeding experiments (described below). Unequivocalassignments for compound 10 were established with 1D nuclear Overhausereffect (NOE) and long range heteronuclear correlation (HETCOR) studies.Deuterium exchange confirmed the presence of hydroxyls at C-15 ofcompound 10 and C-13 of compound 11. Field desorption mass spectrometry(FD-MS) of 2 revealed a molecular weight of 282, consistent withC₁₇H₁₄O₄ (282.2952).

[0231] Earlier studies showed that the polyketide backbone of 2 (Bartel,P. L. et al. J. Bacteriol. (1990) 172:4816) (and by inference, 1) isderived from iterative condensations of 8 acetate residues with a singleketoreduction at C-9. It may also be argued that nanaomycin (8) arisesfrom an identical carbon chain backbone. Therefore, it is very likelythat nanaomycin is a product of the fren PKS genes in S. roseofulvus.Regiospecificity of the first cyclization leading to the formation of 1is guided by the position of the ketoreduction, whereas that of thesecond cyclization is controlled by the act cyclase (Zhang, H. L. et al.J. Org. Chem. (1990) 55:1682).

[0232] In order to trace the carbon chain backbone of RM18 (10), in vivofeeding experiments using [1,2-¹³C₂] acetate were performed onCH999/pRM18, followed by NMR analysis of labelled RM18 (10). The ¹³Ccoupling data (summarized in Table 3) indicate that the polyketidebackbone of RM18 (10) is derived from 9 acetate residues, followed by aterminal decarboxylation (the C-2 ¹³C resonance appears as an enhancedsinglet), which presumably occurs non-enzymatically. Furthermore, theabsence of a hydroxyl group at the C-9 position suggests that aketoreduction occurs at this carbon. Since these two features would beexpected to occur in the putative frenolicin (7) backbone, the resultssuggest that, in addition to synthesizing nanaomycin, the fren PKS genesare responsible for the biosynthesis of frenolicin in S. roseofulvus.This appears to be the first unambiguous case of a PKS with relaxedchain length specificity. However, unlike the putative backbone offrenolicin, the C-17 carbonyl of RM18 (10) is not reduced. This couldeither reflect the absence from pRM18 of a specific ketoreductase,dehydratase, and an enoylreductase (present in the fren gene cluster inS. roseofulvus), or it could reflect a different origin for carbons15-18 in frenolicin.

[0233] Regiospecificity of the first cyclization leading to theformation of RM18 (10) is guided by the position of the ketoreduction;however the second cyclization occurs differently from that in 7 or 1,and is similar to the cyclization pattern observed in RM20 (9), adecaketide produced by the tcm PKS, as described above. Therefore, as inthe case of RM20 (9), it could be argued that the act cyclase cannotcatalyze the second cyclization of the RM18 precursor, and that itssubsequent cyclizations, which presumably occur non-enzymatically, aredictated by temporal differences in release of different portions of thenascent polyketide chain into an aqueous environment. In view of theability of CH999/pRM18 (and CH999/pRM34) to produce 1, one can rule outthe possibility that the cyclase cannot associate with the fren PKS(KS/AT, CLF, and ACP). A more likely explanation is that the act cyclasecannot recognize substrates of altered chain lengths. This, would alsobe consistent with the putative biosynthetic scheme for RM20 (9).

[0234] A comparison of the product profiles of the hybrid synthasesreported in Table 2 with analogous hybrids between act and tcm PKScomponents (Table 1) support the hypothesis that the ORF2 product is thechain length determining factor (CLF). Preparation of compounds 9, 10and 11 via cyclization of enzyme-bound ketides is schematicallyillustrated in FIG. 8.

EXAMPLE 5 The Role of tcmJ and tcmN in Polyketide Synthesis

[0235] To evaluate the specific catalytic roles of the PKS enzymesencoded by tcmJ and tcmN, tcmJ and tcmN were expressed in the presenceof additional act and tcm PKS components in the S. coelicolor CH999host-vector system described in Examples 1 through 3. The isolation ofthree novel polyketides from these genetic constructs has allowed theassignment of two distinct catalytic functions to tcmN.

[0236] The series of recombinant gene clusters shown in Table 5 wasconstructed. Each plasmid contained either tcmJ, tcmN, or both inaddition to the minimal PKS genes responsible for the biosynthesis of 16(act) or 20 (tcm) carbon backbones. Half of the plasmids also containedthe gene encoding the act ketoreductase (KR, actIII), which catalyzesketoreduction at the C-9 position of the nascent polyketide backbone.The plasmids were introduced by transformation into S. coelicolor CH999.The major polyketides produced by the transformed strains were isolatedand structurally characterized using a combination of NMR, isotopiclabelling and mass spectroscopy experiments. All of the polyketidesisolated have been previously structurally characterized with theexception of the novel polyketides RM77 (19) (FIG. 14), RM80 (20), andRM80b (21) (FIG. 15).

[0237] A comparative analysis of the cyclization patterns of thesemolecules, together with those reported earlier, reveals two functionsfor tcmN. The first can be illustrated by differences in the proposedpathways for RM77 (19; produced by the act minimal PKS+tcmN; pRM77) andSEK4 (12; produced by the act minimal PKS alone; pSEK24). As shown inFIG. 14, tcmN influences the regiospecificity of the cyclization of thefirst ring. In SEK4 (12), an intramolecular aldol condensation occursbetween the C-7 carbonyl and the C-12 methylene. In contrast, a similarreaction occurs between the C-9 carbonyl and the C-14 methylene in RM77(19); this represents a shift of one acetate unit in the polyketidebackbone. Thus, while earlier results indicated that the course of thisreaction is primarily controlled by the minimal PKS, RM77 (19) clearlyillustrates the effect of tcmN on the act minimal PKS, which otherwiseexclusively catalyzes C-7/C-12 cyclizations in the absence of tcmN. Theabsence of any significant amount of SEK15 (13) or other C-7/C-12cyclized molecules in CH999/pRM80 and CH999/pRM81 also supports theconclusion that regiospecificity of the first aldol condensation can becontrolled by enzymes downstream of the minimal PKS.

[0238] An important consequence of the designation the tcmN function isthe temporal relationship between the catalytic ketoreduction andcyclization of the first ring. In all naturally occurring andrecombinant polyketides undergoing a C-9 ketoreduction studied to date,initial cyclization occurs between carbons 7 and 12. Therefore, theinability of strains expressing tcmN to produce significant quantitiesof a polyketide with a C-9/C-14 cyclization in the presence of the actKR (pRM71, pRM72, pRM74, pRM75; Table 5) indicates that ketoreductionoccurs prior to formation of the first ring (FIG. 14).

[0239] The second function of tcmN is apparent from comparison betweenthe proposed cyclization pathways of RM80 (20; produced by the tcmminimal PKS+tcmN; pRM80) and SEK15b (16; produced by the tcm minimal PKSalone; pSEK33). Production of these two molecules is mutually exclusivein these strains. As seen in FIG. 15, the regiospecificities of thefirst and second intramolecular aldol condensations in both moleculesare identical. However, in SEK15b (16) the third ring forms via an aldolcondensation between C-6 and C-19, whereas in RM80 (20) it forms viahemiketalization between C-15 and C-19. The difference in these twocyclization pathways can be attributed to enolization of the C-15carbonyl in RM80 (20), but not in SEK15b (16). This is reminiscent ofthe related polyketides SEK34 (22) and mutactin (6), shunt products fromthe early stages of actinorhodin biosynthesis which led to thehypothesis that the act aromatase (ARO) catalyzes the enolization of theC-11 carbonyl. Therefore, it is not surprising that tcmN, a homolog ofthe act ARO, should catalyze the same reaction; however, thespecificities of the two proteins differ. Whereas the act ARO actson-the first ring, tcmN appears to act on the second ring.

[0240] TcmN provides an additional tool for the design and biosynthesisof novel polyketides through the genetic manipulation of PKSs. RM77 (19)represents the first example of a 16-carbon polyketide with anengineered first cyclization different from that of the expected“natural” one. Therefore, it is likely that other heterologous PKScomplexes containing tcmN (or homologs) along with various minimal PKSswill produce polyketides of different chain length with the alternativefirst cyclization. This biosynthetic degree of freedom may be limited tounreduced molecules.

EXAMPLE 6 Rationally Designed Aromatic Polyketides

[0241] All identified gene clusters for actinomycete aromaticpolyketides contain a set of three genes encoding a so-called ‘minimalPKS’ which consists of a ketosynthase (KS), which also carries aputative acyltransferase (AT) domain, a chain length factor (CLF), andan acyl carrier protein (ACP) (FIG. 1). A 16-carbon molecule, forexample SEK4 (12), can be synthesized from the act minimal PKS alone. Inorder to produce the C-9 reduced analogue of SEK4, SEK34 (22) (FIG. 16),two additional activities are needed: a ketoreductase (KR) and anaromatizing subunit (ARO) (compare the genes present on pSEK24 andpSEK34; Table 6). The following experiments were designed to determinewhether analogous pairs of molecules could be generated from backbonesof alternative chain length, for example, 20 carbons using a suitablecombination of a minimal PKS, a KR, and an ARO.

[0242] The tcm minimal PKS (on pSEK33; Table 6) is both necessary andsufficient for synthesis of an unreduced 20 carbon backbone (McDaniel,R. et al. Proc. Natl. Acad. Sci. USA (1994) 91:11542-11546), which formsSEK15 (13). In addition, the act KR can reduce the C-9 carbonyl on sucha backbone to a hydroxyl, which is subsequently lost upon spontaneousaromatization of the first carbocyclic ring (Fu, H. et al. J. Am. Chem.Soc. (1994) 116:4166-4170). Aromatization of the reduced ring, incontrast, requires an ARO (McDaniel, R. et al. J. Am. Chem. Soc. (1994)116:10855-10859). However, the act ARO cannot aromatize 20-carbon chains(McDaniel, R., et al. Science (1994) 262:1546-1550; McDaniel et al.Proc. Natl. Acad. Sci. USA (1994), supra). Furthermore, the tcm PKScluster (which lacks a KR gene) does not appear to encode a first ringARO which would be a suitable candidate. Accordingly, an ARO genehomologous to the one in the act cluster was chosen from the genecluster that encodes the PKS for the 20-carbon polyketide griseusin(gris) (Yu, T. -W. et al. J. Bacteriol. (1994) 176:2627-2534).

[0243] The plasmid pSEK43 (Table 6), containing the tcm minimal PKS, theact KR, and the gris ARO, was constructed and introduced into the CH999host. Analysis of the transformed strain revealed the anticipatedpolyketide SEK43 (23), whose structure was determined by NMR, massspectroscopy, and isotopic labelling studies.

[0244] The biosynthesis of SEK43 (23) (FIG. 17) reaffirms the conclusionthat the act ARO and its homologues aromatize the first ring (McDanielet al. J. Am. Chem. Soc. (1994), supra). Without a functional ARO, thetcm minimal PKS and act KR (pSEK23; Table 6) produce RM20b (14) (FIG.16), which contains a non-aromatized first ring. Replacement of the tcmminimal PKS in pSEK43 with either the act or fren minimal PKSs (pSEK41and pSEK42; Table 6) resulted in production of the 16-carbon aromatizedcompound SEK34 (22), demonstrating that the gris ARO can also recognizeshorter carbon chains. It was unexpected, however, that a corresponding18-carbon polyketide was not detected in the construct containing thefren minimal PKS, which has been shown to. synthesize both 18- and16-carbon chains (McDaniel et al. J. Am. Chem. Soc. (1993), supra). Thisis probably due to decomposition of the molecule, since CH999/pSEK42produced small quantities of an uncharacterized molecule not present inCH999/pSEK34. More significantly, evidence for an aromatized 18-carbonintermediate is described below.

[0245] A second test of the concept of rational design arose from theprevious isolation of the 16-carbon polyketide DMAC (28) (FIG. 16). ThePKS subunits required for DMAC (28) biosynthesis are a “16-carbon”minimal PKS, a KR, and suitable ARO and CYC components (pRM5; Table 6).CYC catalyzes cyclization of the second ring between carbons 4 and 15,leading eventually to the formation of an anthraquinone (McDaniel et al.J. Am. Chem. Soc. (1994), supra). These observations suggested that ananalogous anthraquinone, with 18 carbons, could be generated. To achievethis, the plasmid pSEK26 (Table 6) containing the fren minimal PKS withthe act KR, the fren ARO, and the act CYC was constructed. The frenminimal PKS and act KR were selected for their ability to produce an18-carbon, C-9 reduced backbone (McDaniel et al. J. Am. Chem. Soc.(1993), supra; McDaniel et al. Proc. Natl. Acad. Sci. USA (1994),supra). The fren ARO was chosen since the act ARO cannot aromatize18-carbon chains (McDaniel et al. J. Am. Chem. Soc. (1993), supra;McDaniel et al. Proc. Natl. Acad. Sci. USA (1994), supra).

[0246] Introduction of the plasmid into CH999 resulted in the productionof both DMAC (28) and SEK26 (24). The latter is a novel 18-carbonanthraquinone whose structure was confirmed by NMR, mass spectroscopy,and isotopic labelling studies. Formation of SEK26 (24) (FIG. 17) occursthrough a second ring cyclization at C5/C14 presumably catalyzed by theact CYC. The production also of DMAC (28) is consistent with the relaxedchain length specificity of the fren minimal PKS (McDaniel et al. J. Am.Chem. Soc. (1993), supra).

[0247] In order to evaluate further the specificity of ARO and CYCsubunits towards carbon chains of various lengths, several other PKScombinations were constructed (Table 6). For example, pSEK25 and pSEK26demonstrate that the fren ARO can aromatize both 16- and 18-carbonchains. However, the fren ARO cannot handle 20-carbon chains; insteadthe combination of the fren ARO with the tcm minimal PKS, act KR, andact CYC (pSEK27) resulted in biosynthesis of RM20b (14), thenon-aromatized 20-carbon polyketide (FIG. 16). As expected, replacingthe fren ARO with the gris ARO (pRM51) in pSEK26 yielded DMAC (28) andSEK26 (24). However, attempts to generate a 20-carbon reduced polyketidewith a C-5/C-14 second ring cyclization were unsuccessful; replacing thefren minimal PKS in pRM51 with the tcm minimal PKS (pRM52) resulted inproduction of SEK43 (23), indicating that the act CYC cannot cyclize20-carbon chains. Finally, the plasmids pSEK44-47, pRM51, and pRM52(Table 6), all lacking KRs, failed to cause production of polyketidesdifferent from those produced by the minimal PKS alone (McDaniel et al.Proc. Natl. Acad. Sci. USA (1994), supra), despite the presence of AROand CYC components. This is consistent with previous observations thatARO and CYC subunits do not alter the biosynthetic pathways of unreducedpolyketides (McDaniel et al. Proc. Natl. Acad. Sci. USA (1994), supra;Fu, H., McDaniel, R., Hopwood, D. A. & Khosla, C. Biochemistry33:9321-9326 (1994)).

EXAMPLE 7 Construction and Analysis of Modular Polyketide Synthases

[0248] Expression plasmids containing recombinant modular DEBS PKS geneswere constructed by transferring DNA incrementally from atemperature-sensitive “donor” plasmid, i.e., a plasmid capable ofreplication at a first, permissive temperature and incapable ofreplication at a second, non-permissive temperature, to a “recipient”shuttle vector via a double recombination event, as depicted in FIG. 18.pCK7 (FIG. 12), a shuttle plasmid containing the complete eryA genes,which were originally cloned from pS1 (Tuan et al. (1990) Gene 90:21),was constructed as follows. A 25.6 kb SphI fragment from pS1 wasinserted into the SphI site of pMAK705 (Hamilton et al. (1989) J.Bacteriol. 171:4617) to give pCK6 (Cm^(R)), a donor plasmid containingeryAII, eryAIII, and the 3′ end of eryAI. Replication of thistemperature-sensitive pSC101 derivative occurs at 30° C. but is arrestedat 44° C. The recipient plasmid, pCK5 (Ap^(R), Tc^(R)), includes a 12.2kb eryA fragment from the eryAI start codon (Caffrey et al. (1992) FEBSLett. 304:225) to the XcmI site near the beginning of eryAII, a 1.4 kbEcoRI-BsmI pBR322 fragment encoding the tetracycline resistance gene(Tc), and a 4.0 kb NotI-EcoRI fragment from the end of eryAIII. PacI,NdeI, and ribosome binding sites were engineered at the eryAI startcodon in pCK5. pCK5 is a derivative of pRM5 (McDaniel et al. (1993),supra). The 5′ and 3′ regions of homology (FIG. 18, striped and unshadedareas) are 4.1 kb and 4.0 kb, respectively. MC1061 E. coli wastransformed (see, Sambrook et al., supra) with pCK5 and pCK6 and.subjected to carbenicillin and chloramphenicol selection at 30° C.Colonies harboring both plasmids (Ap^(R), Cm^(R)) were then restreakedat 44° C. on carbenicillin and chloramphenicol plates. Only cointegratesformed by a single recombination event between the two plasmids wereviable. Surviving colonies were propagated at 30° C. under carbenicillinselection, forcing the resolution of the cointegrates via a secondrecombination event. To enrich for pCK7 recombinants, colonies wererestreaked again on carbenicillin plates at 44° C. Approximately 20% ofthe resulting colonies displayed the desired phenotype (Ap^(R),Tc^(S),Cm^(S)). The final pCK7 candidates were thoroughly checked viarestriction mapping. A control plasmid, pCK7f, which contains aframeshift error in eryAI, was constructed in a similar manner. pCK7 andpCK7f were transformed into E. coli ET12567 (MacNeil (1988) J.Bacteriol. 170:5607) to generate unmethylated plasmid DNA andsubsequently moved into Streptomyces coelicolor CH999 using standardprotocols (Hopwood et al. (1985) Genetic manipulation of Streptomyces. Alaboratory manual. The John Innes Foundation: Norwich).

[0249] Upon growth of CH999/pCK7 on R2YE medium, the organism producedabundant quantities of two polyketides (FIG. 20). The addition ofpropionate (300 mg/L) to the growth medium resulted in approximately atwo-fold increase in yield of polyketide product. Proton and ¹³C NMRspectroscopy, in conjunction with propionic-1-¹³C acid feedingexperiments, confirmed the major product as 6dEB (17) (>40 mg/L). Theminor product was identified as 8,8a-deoxyoleandolide (18) (>10 mg/L),which apparently originates from an acetate starter unit instead ofpropionate in the 6dEB biosynthetic pathway. ¹³C₂ sodium acetate feedingexperiments confirmed the incorporation of acetate into (18). Three highmolecular weight proteins (>200 kDa), presumably DEBS1, DEBS2, and DEBS3(Caffrey et al. (1992) FEBS Lett. 304:225), were also observed in crudeextracts of CH999/pCK7 via SDS-polyacrylamide gel electrophoresis. Nopolyketide products were observed from CH999/pCK7f. The inventors herebyacknowledge support provided by the American Cancer Society (IRG-32-34).

EXAMPLE 8 Manipulation of Macrolide Ring Size by Directed Mutagenesis ofDEBS

[0250] In order to investigate the relationship between structure andfunction in modular PKSs and to apply this knowledge towards therational and stochastic design of novel polyketides, a host-vectorexpression system was designed to study DEBS (Kao, C. M. et al. Science(1994) 265:509-512). Using this expression system, the expression ofDEBS1 alone, in the absence of DEBS2 and DEBS3, resulted in theproduction of (2R,3S,4S,5R)-2,4-dimethyl-3,5-dihydroxy-n-heptanoic acidδ-lactone (“the heptanoic acid δ-lactone” (25)) (1-3 mg/L), the expectedtriketide product of the first two modules (FIG. 21A) (Kao, C. M. et al.J. Am. Chem. Soc. (1994) 116:11612-11613). The synthesis of theheptanoic acid δ-lactone (25) provided further biochemical evidence forthe modular PKS model of Katz and coworkers (Donadio, S. et al. Science(1991), supra) and showed that a thioesterase is not essential forrelease of a triketide from the enzyme complex.

[0251] In this Example the role of the thioesterase (TE) domain in DEBSwas analyzed by constructing two additional deletion mutant PKSs thatconsist of different subsets of the DEBS modules and the TE. The firstPKS contained DEBS1 fused to the TE, whereas the second PKS included thefirst five DEBS modules with the TE; plasmids pCK12 and pCK15 containedthe genes encoding the bimodular (“1+2+TE”) and pentamodular(“1+2+3+4+5+TE”) PKSs.

[0252] The 1+2+TE PKS contained a fusion of the carboxy-terminal end ofthe acyl carrier protein of module 2 (ACP-2) to the carboxy-terminal endof the acyl carrier protein of module 6 (ACP-6). Thus ACP-2 isessentially intact in this PKS and is followed by the amino acidsequence naturally found between ACP-6 and the TE (FIG. 21B). PlasmidpCK12 contained eryA DNA originating from pS1 (Tuan, J. S. et al. Gene(1990) 90:21). pCK12 is identical to pCK7 (Kao et al. Science (1994),supra) with the exception of a deletion between the carboxy-terminalends of ACP-2 and ACP-6. The fusion occurs between residues L3455 ofDEBS1 and Q2891 of DEBS3. An SpeI site is present between these tworesidues so that the DNA sequence at the fusion is CTCACTAGTCAG.

[0253] The 1+2+3+4+5+TE PKS contained a fusion 76 amino acids downstreamof the β-ketoreductase of module 5 (KR-5) and five amino acids upstreamof ACP-6. Thus, the fusion occurs towards the carboxy-terminal end ofthe non-conserved region between KR-5 and ACP-5, and the recombinantmodule 5 was essentially a hybrid between the wild type modules 5 and 6(FIG. 22). Plasmid pCK15 contained eryA DNA originating from pS1 (Tuanet al. Gene (1990), supra). pCK15 is a derivative of pCK7 (Kao et al.Science (1994), supra) and was constructed using an in vivorecombination strategy described earlier (Kao et al. Science (1994),supra). pCK15 is identical to pCK7 with the exceptions of a deletionbetween KR-5 and ACP-6, which occurs between residues G1372 and A2802 ofDEBS3, and the insertion of a blunted a SalI fragment containing akanamycin resistance gene (Oka A. et al. J. Mol. Biol. (1981) 147:217)into the blunted HindIII site of pCK7. An arginine residue is presentbetween G1372 and A2802 so that the DNA sequence at the fusion isGGCCGCGCC.

[0254] Plasmids pCK12 and pCK15 were introduced into S. coelicolor CH999and polyketide products purified from the transformed strains accordingto methods previously described (Kao et al. Science (1994), supra).

[0255] CH999/pCK12 produced the heptanoic acid δ-lactone (25) (20 mg/L)as determined by ¹H and ¹³C NMR spectroscopy. This triketide product isidentical to that produced by CH999/pCK9, which expresses the unmodifiedDEBS1 protein alone (Kao J. Am. Chem. Soc. (1994), supra. However,CH999/pCK12 produced the heptanoic acid δ-lactone (25) in significantlygreater quantities than CH999/pCK9 (>10 mg/L vs. ˜1 mg/L), indicatingthe ability of the TE to catalyze thiolysis of a triketide chainattached to the ACP domain of module 2. CH999/pCK12 also producedsignificant quantities of a novel analog of(2R,3S,4S,5R)-2,4-dimethyl-3,5-dihydroxy-n-hexanoic acid δ-lactone (“thehexanoic acid δ-lactone (26)) (10 mg/L), that resulted from theincorporation of an acetate start unit instead of propionate. This isreminiscent of the ability of CH999/pCK7, which expresses intact DEBS,to produce 8,8a-deoxyoleandolide (18) in addition to 6dEB (17) (Kao etal. Science (1994), supra).

[0256] Since the hexanoic acid δ-lactone (26) was not detected inCH999/pCK9, its facile isolation from CH999/pCK12 provides additionalevidence for the increased turnover rate of DEBS1 due to the presence ofthe TE. In other words. the TE can effectively recognize an intermediatebound to a “foreign” module that is four acyl units shorter than itsnatural substrate, 6dEB (17). However, since the triketide products canprobably cyclize spontaneously into the heptanoic acid δ-lactone (25)and the hexanoic acid δ-lactone (26) under typical fermentationconditions (pH 7), it is not possible to discriminate between abiosynthetic model involving enzyme-catalyzed lactonization and oneinvolving enzyme-catalyzed hydrolysis followed by spontaneouslactonization. Thus, the ability of the 1+2+TE PKS to recognize the C-5hydroxyl of a triketide as an incoming nucleophile is unclear.

[0257] The second recombinant strain, CH999/pCK15, produced abundantquantities of (8R,9S)-8,9-dihydro-8-methyl-9-hydroxy-10-deoxymethonolide(“the 10-deoxymethonolide (27); FIG. 22) (10 mg/L), demonstrating thatthe pentamodular PKS is active. The 10-deoxymethonolide (27) wascharacterized using ¹H and ¹³C NMR spectroscopy of natural abundance and¹³C-enriched material, homonuclear correlation spectroscopy (COSY),heteronuclear correlation spectroscopy (HETCOR), mass spectrometry, andmolecular modeling. The 10-deoxymethonolide (27) is an analog of10-deoxymethonolide (Lambalot, R. H. et al. J. Antibiotics (1992)45:1981-1982), the aglycone of the macrolide antibiotic methymycin. Theproduction of the 10-deoxymethonolide (27) by a pentamodular enzymedemonstrates that active site domains in modules 5 and 6 in DEBS can bejoined without loss of activity. If this proves to be a general featureof the multimodular proteins that constitute modular PKSS, then anystructural model for module assembly must account for the fact thatindividual modules as well as active sites are independent entitieswhich do not depend on association with neighboring modules to befunctional. Most remarkably, the 12-membered lactone ring, formed byesterification of the terminal carboxyl with the C-11 hydroxyl of thehexaketide product, indicated the ability of the 1+2+3+4+5+TE PKS, andpossibly the TE itself, to catalyze lactonization of a polyketide chainone acyl unit shorter than the natural product of DEBS, 6dEB (17)Indeed, the formation of the 10-deoxymethonolide (27) may mimic thebiosynthesis of the closely related 12-membered hexaketide macrolide,methymycin, which frequently occurs with the homologous 14-memberedheptaketide macrolides, picromycin and/or narbomycin (Cane, D. E. et al.J. Am. Chem. Soc. (1993)-115:522-566). A modular PKS such as DEBS couldthus be used to generate a wide range of macrolactones with shorter aswell as longer chain lengths. The latter products would require theintroduction of additional heterologous modules into DEBS.

[0258] The construction of the 1+2+3+4+5+TE PKS resulted in thebiosynthesis of a previously uncharacterized 12-membered macrolactonethat closely resembles, but is distinct from, the aglycone of abiologically active macrolide. The apparent structural and functionalindependence of active site domains and modules as well as relaxedlactonization specificity suggest the existence of many degrees offreedom for manipulating these enzymes to produce new modular PKSs.Libraries of new macrolides can be generated by altering the associationof active site domains and entire modules, the subset of reductivedomains within each module, the activity of the TE, and possibly evendownstream modification reactions such as hydroxylation andglycosylation. Such libraries could prove to be rich sources of newleads for drug discovery

[0259] Thus, novel polyketides, as well as methods for recombinantlyproducing the polyketides, are disclosed. Although preferred embodimentsof the subject invention have been described in some detail, it isunderstood that obvious variations can be made without departing fromthe spirit and the scope of the invention as defined by the appendedclaims. TABLE 1 Backbone ORF1 ORF2 ORF3 Major Carbon Plasmid (KS/AT)(CLDF) (ACP) Product(s) Length pRM5  act act act 1, 2 16 pRM7  gra actact 1, 2 16 pRM12 act gra act 1, 2 16 pRM22 act act gra 1, 2 16 pRM10tcm act act 1, 2 16 pRM15 act tcm act NP — pRM20 tcm tcm act 9 20 pRM25act act tcm 1, 2 16 pRM35 tcm act tcm NP — pRM36 act tcm tcm NP — pRM37tcm tcm tcm 9, 14, 15 20 pSEK15 tcm tcm tcm 13, 16 20 pSEK33 tcm tcm act13, 16 20

[0260] TABLE 2 ORF1 ORF2 ORF3 Major Plasmid (KS/AT) (CLDF) (ACP)Product(s) pRM5  act act act 1, 2 pRM8  fren act act 1, 2 pRM13 act frenact NP pRM23 act act fren 1, 2 pRM18 fren fren act 1, 2, 10, 11 pRM32fren act fren NP pRM33 act fren fren NP pRM34 fren fren fren 0001, 2,10, 11

[0261] TABLE 3 ¹H(400 MHz) and ¹³C(100 MHz) NMR data from RM18(10) andRM18b(11) RM18 RM18b ¹Hδ(ppm) ¹Hδ(ppm) carbon^(α) ¹³Cδ(ppm) (J_(CC)(Hz))(m, J_(HH)(HZ), area)) carbon^(α) ¹³Cδ(ppm) (m, J_(HH)(HZ), area)) 229.6 NC^(b) 2.2(s, 3H) 3 203.7 37.7 4 47.0 36.9 3.6(s, 2H) 2 18.8 2.1(s,3H) 5 149.6 77.2 3 152.3 6 106.7 77.4 6.2(s, 1H) 4 104.0 6.1(s, 1H) 7129.1 61.9 5 130.0 8 114.4 62.1 6.7(d, 7.2, 1H) 6 113.5 6.7(d, 7.0) 9130.1 58.9 7.3(dd, 8.4, 7.4, 1H) 7 130.1 7.3(dd, 7.1, 8.7, 1H) 10 120.659.2 7.6(d, 8.9, 1H) 8 120.1 7.6(d, 8.6, 1H) 11 132.7 56.0 9 132.8 12116.7 55.7 10 116.6 13 155.6 74.7 11 155.9 14 98.4 74.9 12 98.2 15 158.869.6 6.4(s, 1H) 13 159.1 6.4(s, 1H) 16 113.6 69.3 11.2(s, 1OH) 14 113.811.2(s, 1OH) 17 201.7 41.9 15 201.7 18 32.4 41.7 2.5(s, 3H) 16 32.42.5(s, 3H)

[0262] TABLE 4 ¹H and ¹²C NMR data for SEK4 (12) and SEK15 (13)^(a) SEK4(12) SEK15 (13) carbon^(b) ¹³Cδ(ppm) J_(CC)(Hz) ¹Hδ(ppm) carbon¹³Cδ(ppm) J_(CC)(Hz) ¹Hδ(ppm) 1 165.4 78.8 11.60(s, 1OH) 1 164.0 79.112.20(s, 1OH) 2 88.2 79.8 6.26(d, J = 2.28 Hz, 1H) 2 88.2 79.4 6.20(d, J= 1.88 Hz, 1H) 3 170.5 55.3 3 172.8 57.9 4 111.3 61.3 6.33(d, J = 2.24Hz, 1H) 4 101.8 53.9 6.20(d, J = 1.88 Hz, 1H) 5 163.8 51.0 5 163.1 50.46 37.6 50.8 4.07(d, J = 15.7 Hz, 1H) 6 36.7 50.8 1.90(s, 2H) 4.16(d, J =16.0 Hz, 1H) 7 138.6 60.7 7 135.4 60.7 8 102.9 60.9 5.66(d, J = 1.6 Hz,1H) 8 109.1 61.7 5.66 (s, 1H) 9 161.9 71.9 10.50(s, 1OH) 9 159.8 66.2 10100.6 70.9 5.19(d, J = 1.96 Hz, 1H) 10 101.6 66.5 5.08 (s, 1H) 11 162.960.8 11 157.4 67.3 12 112.9 61.6 12 121.1 67.6 13 191.1 39.1 13 200.358.1 14 49.3 39.9 2.54(d, J = 15.9 Hz, 1H) 14 117.2 58.6 4.92(d, J =16.0 Hz, 1H) 15 99.6 46.6 6.90(s, 1OH) 15 163.6 68.5 16 27.5 46.81.56(s, 3H) 16 100.6 68.0 6.08 (s, 1H) 17 162.2 62.6 18 111.0 62.0 6.12(s, 1H) 19 141.9 43.3 20 21.1 42.7 1.86 (s, 3H)

[0263] TABLE 5 Minimal Major Plasmid PKS* KR tcmJ, N Product(s) pSEK21act act — 6 pRM70 act act J 6 pRM71 act act N 6 pRM72 act act J, N 6pSEK23 tcm act 10 pRM73 tcm act J 10 pRM74 tcm act N 10 pRM75 tcm act J,N 10 pSEK24 act — — 12 pRM76 act — J 12 pRM77 act — N 19 pRM78 act — J,N 19 pSEK33 tcm — — 13, 16 pRM79 tcm — J 13, 16 pRM80 tcm — N 20, 21pRM81 tcm — J, N 20, 21

[0264] TABLE 6 Minimal Major Plasmid PKS* KR ARO CYC Product pSEK24 act— — — 12 pSEK34 act act act — 22 pRM5  act act act act 28 pSEK33 tcm — —— 13 pSEK23 tcm act — — 14 pSEK41 act act gris — 22 pSEK42 fren act gris— 22 pSEK43 tcm act gris — 23 pSEK25 act act fren act 28 pSEK26 fren actfren act 28/24 pSEK27 tcm act fren act 14 pRM51 fren act gris act 28/24pRM52 tcm act gris act 23 pSEK44 act — gris — 12 pSEK45 tcm — gris — 13pSEK46 act — fren act 12 pSEK47 tcm — fren act 13 pRM53 act — gris act12 pRM54 tcm — gris act 13

1. A method for modifying the acyltransferase active site of a module ofa modular polyketide synthase (PKS) comprising replacing a firstacyltransferase active site of a PKS with a second acyltransferaseactive site of the PKS, wherein the PKS is from Saccharopolysporaerythraea.
 2. A method for modifying the acyltransferase domain of amodule of a modular polyketide synthase (PKS) comprising modifying afirst region encoding a first acyltransferase of a PKS by replacing thefirst region with a second region encoding a second acyltransferase froma different region of the PKS, wherein the PKS is from Saccharopolysporaerythraea.